Methods for the improvement of product yield and production in a microorganism through the addition of alternate electron acceptors

ABSTRACT

The present invention provides for novel metabolic pathways to reduce or eliminate glycerol production and increase product formation. More specifically, the invention provides for a recombinant microorganism comprising a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source, such as lignocellulose, to a product, such as ethanol, wherein the one or more native and/or heterologous enzymes is activated, upregulated, or downregulated. The invention also provides for a recombinant microorganism comprising one or more heterologous enzymes that function to regulate glycerol synthesis and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International ApplicationNumber PCT/US2012/032443, filed Apr. 5, 2012, which claims the benefitof U.S. Provisional Application No. 61/472,085, filed Apr. 5, 2011,which are incorporated by reference herein in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2608_(—)0550001_SEQIDListing.ascii.txt; Size:437,882 bytes; and Date ofCreation: Oct. 4, 2013) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

The conversion of biomass, such as corn, sugarcane or other energycrops, as well as simple sugars, to ethanol is routinely completedthrough the use of yeast fermentation. However, during yeast metabolisma major byproduct of fermentation is glycerol. Glycerol is formed duringanaerobic growth as a way for the yeast to balance its redox state andregenerate NAD⁺ used as a cofactor during glycolysis. It has been shownthat the function of glycerol is likely not as a metabolite itself butrather as an electron sink capturing electrons allowing furthergrowth-linked metabolism to continue. As glycerol is a byproduct withlow value, it can be an undesirable by-product of fermentation. It wouldbe beneficial to reduce or eliminate this by-product and further directmore carbon towards desired end-products, such as ethanol.

Several strategies are available in the art for the conversion ofglycerol to higher value products though biochemical or other means, butrelatively little has been demonstrated for the removal or reduction ofglycerol and improvement of overall sugar yield to ethanol or otherdesired end-products of metabolism. Through engineering of alternatepathways, potentially with the simultaneous reduction or deletion of theglycerol pathway, alternate or replacement electron acceptors for theregeneration of NAD⁺ can be used during yeast metabolism. Such alternateor replacement electron acceptors could be molecules such as formate orhydrogen.

The elimination of glycerol synthesis genes has been demonstrated butremoval of this pathway completely blocked anaerobic growth of theyeast, preventing useful application during an industrial process.Ansell, R., et al., EMBO J. 16:2179-87 (1997); Pahlman, A-K., et al., J.Biol. Chem. 276:3555-63 (2001); Guo, Z P., et al., Metab. Eng. 13:49-59(2011). Other methods to bypass glycerol formation require theco-utilization of additional carbon sources, such as xylose or acetate,to serve as electron acceptors. Lidén, G., et al., Appl. Env. Microbiol.62:3894-96 (1996); Medina, V. G., et al., Appl. Env. Microbiol.76:190-195 (2010). By incorporating a formate pathway as an alternateelectron acceptor, glycerol formation can be bypassed and ethanol yieldcan be increased. The engineering of a pyruvate formate lyase from E.coli, which is capable of converting pyruvate to formate, has been doneto increase formate production. Waks, Z., and Silver, P. A., Appl. Env.Microbiol. 75:1867-1875 (2009). Formate engineering in Waks and Silverwas done, however, to provide a source of formate in S. cerevisiae forthe production of hydrogen by a secondary microorganism, E. coli. Waksand Silver did not combine formate production with the removal ofglycerol formation, and the use of formate as an alternate electronacceptor for the reduction of glycerol was not proposed or evaluated.Thus, despite prior efforts to bypass and/or eliminate glycerolproduction, there exists a need for the engineering of alternate orreplacement electron acceptors in a cell to direct more carbon towardsdesired end-products, such as ethanol.

The importance of engineering alternate or replacement electronacceptors is exemplified in the process of corn mash fermentation. About16 billion gallons of corn-based ethanol are produced annually, so evensmall increases in ethanol yield, such as 5-10%, can translate into anextra billion or so gallons of ethanol over current yields. Ethanolproduction from corn mash typically results in glycerol yields rangingfrom 10-12 g/L. See Yang, R. D., et al., “Pilot plant studies of ethanolproduction from whole ground corn, corn flour, and starch,” Fuel AlcoholU.S.A., Feb. 13-16, 1982 (reported glycerol levels to be as high as 7.2%w/w of initial sugar consumed in normal corn mash fermentations orapproximately 1.4 g/100 mL using 20% sugar). By reducing or eliminatingthe glycerol yield in the production of ethanol from corn andre-engineering metabolic processes, increased ethanol yields can beachieved. Additional benefits may be gained in the production of ethanolfrom corn. Corn mash is a nutrient rich medium, in some cases containinglipid and protein content that can be >3% of the total fermentationvolume. As a result of the energy contained in these components, evenhigher ethanol yields may be achieved than what is predicted using, forexample, pure sugar. The additional increases can come from themetabolism of lipids or amino acids in the corn mash medium. Therecombinant cells and methods of the invention enable increasing ethanolyields from biomass fermentation by reducing or eliminating glycerol.

BRIEF SUMMARY OF THE INVENTION

The invention is generally directed to the reduction or removal ofglycerol production in a host cell and to the engineering of analternate electron acceptor for the regeneration of NAD⁺.

One aspect of the invention relates to a recombinant microorganismcomprising: a deletion of one or more native enzymes that function toproduce glycerol and/or regulate glycerol synthesis; and one or morenative and/or heterologous enzymes that function in one or moreengineered metabolic pathways to convert a carbohydrate source toethanol, wherein said one or more native and/or heterologous enzymes isactivated, upregulated or downregulated. In some embodiments, therecombinant microorganism produces less glycerol than a controlrecombinant microorganism without deletion of said one or more nativeenzymes that function to produce glycerol and/or regulate glycerolsynthesis. In some embodiments, the carbohydrate source is biomass. Insome embodiments, the biomass comprises a lignocellulosic materialselected from the group consisting of grass, switch grass, cord grass,rye grass, reed canary grass, mixed prairie grass, miscanthus,sugar-processing residues, sugarcane bagasse, sugarcane straw,agricultural wastes, rice straw, rice hulls, barley straw, corn cobs,cereal straw, wheat straw, canola straw, oat straw, oat hulls, cornfiber, stover, soybean stover, corn stover, forestry wastes, recycledwood pulp fiber, paper sludge, sawdust, hardwood, softwood, agave, andcombinations thereof. In some embodiments, the biomass is corn mash orcorn starch.

In particular aspects, the one or more native enzymes that function toproduce glycerol are encoded by a gpd1 polynucleotide, a gpd2polynucleotide, or both a gpd1 polynucleotide and a gpd2 polynucleotide.In certain embodiments, the recombinant microorganism further comprisesa native and/or heterologous gpd1 polynucleotide operably linked to anative gpd2 promoter polynucleotide. In other aspects, the one or morenative enzymes that function to produce glycerol are encoded by a gpp1polynucleotide, a gpp2 polynucleotide, or both a gpp1 polynucleotide anda gpp2 polynucleotide.

In particular aspects, the one or more native enzymes that function toregulate glycerol synthesis are encoded by an fps1 polynucleotide.

In further aspects, the engineered metabolic pathways compriseconversion of pyruvate to acetyl-CoA and formate. In certainembodiments, pyruvate is converted to acetyl-CoA and formate by apyruvate formate lyase (PFL). In some embodiments, the PFL is ofprokaryotic or eukaryotic origin. In some embodiments, PFL is from oneor more of a Bifidobacteria, an Escherichia, a Thermoanaerobacter, aClostridia, a Streptococcus, a Lactobacillus, a Chlamydomonas, aPiromyces, a Neocallimastix, or a Bacillus species. In some embodiments,PFL is from one or more of a Bacillus licheniformis, a Streptococcusthermophilus, a Lactobacillus plantarum, a Lactobacillus casei, aBifidobacterium adolescentis, a Clostridium cellulolyticum, aEscherichia coli, a Chlamydomonas reinhardtii PflA, a Piromyces sp. E2,or a Neocallimastix frontalis. In one embodiment, PFL is from aBifidobacterium adolescentis.

In additional aspects, the engineered metabolic pathways compriseconversion of acetyl-CoA to ethanol. In certain embodiments, acetyl-CoAis converted to acetaldehyde by an acetaldehyde dehydrogenase andacetaldehyde is converted to ethanol by an alcohol dehydrogenase. Incertain embodiments, acetyl-CoA is converted to ethanol by abifunctional acetaldehyde/alcohol dehydrogenase. In some embodiments,the acetaldehyde dehydrogenase, alcohol dehydrogenase, or bifuntionalacetaldehyde/alcohol dehydrogenase is of prokaryotic or eukaryoticorigin. In one embodiment, acetaldehyde dehydrogenase is from C.phytofermentans. In some embodiments, bifunctional acetaldehyde/alcoholdehydrogenase is from an Escherichia, a Clostridia, a Chlamydomonas, aPiromyces, or a Bifidobacteria species. In some embodiments,bifunctional acetaldehyde/alcohol dehydrogenase is from Escherichiacoli, Clostridium phytofermentans, Chlamydomonas reinhardtii, Piromycessp. E2, or Bifidobacterium adolescentis. In one embodiment, bifunctionalacetaldehyde/alcohol dehydrogenase is from a Bifidobacteriumadolescentis or Piromyces sp. E2.

In further aspects, the recombinant microorganism comprises a deletionof one or more native enzymes encoded by an fdh1 polynucleotide, an fdh2polynucleotide, or both an fdh1 polynucleotide and an fdh2polynucleotide.

In certain embodiments, the carbohydrate source for the recombinantmicroorganism is lignocellulose. In certain embodiments, the recombinantmicroorganism produces ethanol. In certain embodiments, the recombinantmicroorganism produces formate.

In certain embodiments, the recombinant microorganism is selected fromthe group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis,Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenulapolymorphs, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans,Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus,Schizosaccharomyces pombe, Candida albicans, and Schwanniomycesoccidentalis. In one embodiment, the recombinant microorganism isSaccharomyces cerevisiae.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to produce glycerol encoded by both agpd1 polynucleotide and a gpd2 polynucleotide, an engineered metabolicpathway that comprises conversion of pyruvate to acetyl-CoA and formateby a pyruvate formate lyase and an engineered metabolic pathway thatcomprises conversion of acetyl-CoA to ethanol by a bifunctionalacetaldehyde/alcohol dehydrogenase, and the recombinant microorganismfurther comprises a deletion of one or more native enzymes encoded byboth an fdh1 polynucleotide and an fdh2 polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to produce glycerol encoded by both agpp1 polynucleotide and a gpp2 polynucleotide, an engineered metabolicpathway that comprises conversion of pyruvate to acetyl-CoA and formateby a pyruvate formate lyase. In further embodiments, one engineeredmetabolic pathway of the recombinant microorganism converts acetyl-CoAto ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase and therecombinant microorganism further comprises a deletion of one or morenative enzymes encoded by both an fdh1 polynucleotide and an fdh2polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to regulate glycerol synthesis encodedby an fps1 polynucleotide, an engineered metabolic pathway thatcomprises conversion of pyruvate to acetyl-CoA and formate by a pyruvateformate lyase. In further embodiments, one engineered metabolic pathwayof the recombinant microorganism converts acetyl-CoA to ethanol by abifunctional acetaldehyde/alcohol dehydrogenase and the recombinantmicroorganism further comprises a deletion of one or more native enzymesencoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to regulate glycerol synthesis encodedby an fps1 polynucleotide and one or more native enzymes that functionto produce glycerol encoded by both a gpd1 polynucleotide and a gpd2polynucleotide, and an engineered metabolic pathway that comprisesconversion of pyruvate to acetyl-CoA and formate by a pyruvate formatelyase and an engineered metabolic pathway that comprises conversion ofacetyl-CoA to ethanol by a bifunctional acetaldehyde/alcoholdehydrogenase, and the recombinant microorganism further comprises adeletion of one or more native enzymes encoded by both an fdh1polynucleotide and an fdh2 polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to regulate glycerol synthesis encodedby an fps1 polynucleotide, an engineered metabolic pathway thatcomprises conversion of pyruvate to acetyl-CoA and formate by a pyruvateformate lyase and an engineered metabolic pathway that comprisesconversion of acetyl-CoA to ethanol by a bifunctionalacetaldehyde/alcohol dehydrogenase, and the recombinant microorganismfurther comprises a deletion of one or more native enzymes encoded byboth an fdh1 polynucleotide and an fdh2 polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to produce glycerol encoded by both agpd1 polynucleotide and a gpd2 polynucleotide, an engineered metabolicpathway that comprises conversion of pyruvate to acetyl-CoA and formateby a pyruvate formate lyase and an engineered metabolic pathway thatcomprises conversion of acetyl-CoA to ethanol by a bifunctionalacetaldehyde/alcohol dehydrogenase, and the recombinant microorganismfurther comprises a native and/or heterologous gpd1 polynucleotideoperably linked to a native gpd2 promoter polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to produce glycerol encoded by both agpd1 polynucleotide and a gpd2 polynucleotide, and an engineeredmetabolic pathway that comprises conversion of pyruvate to acetyl-CoAand formate by a pyruvate formate lyase and an engineered metabolicpathway that comprises conversion of acetyl-CoA to ethanol by abifunctional acetaldehyde/alcohol dehydrogenase, further comprising anative and/or heterologous gpd1 polynucleotide operably linked to anative gpd2 promoter polynucleotide and a deletion of one or more nativeenzymes encoded by both an fdh1 polynucleotide and an fdh2polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to produce glycerol encoded by both agpd1 polynucleotide and a gpd2 polynucleotide and one or more nativeenzymes that function to regulate glycerol synthesis encoded by an fps1polynucleotide, and an engineered metabolic pathway that comprisesconversion of pyruvate to acetyl-CoA and formate by a pyruvate formatelyase and an engineered metabolic pathway that comprises conversion ofacetyl-CoA to ethanol by a bifunctional acetaldehyde/alcoholdehydrogenase, further comprising a native and/or heterologous gpd1polynucleotide operably linked to a native gpd2 promoter polynucleotide.

In certain embodiments, the recombinant microorganism comprises one ormore native enzymes that function to produce glycerol encoded by both agpd1 polynucleotide and a gpd2 polynucleotide and one or more nativeenzymes that function to regulate glycerol synthesis encoded by an fps1polynucleotide, and an engineered metabolic pathway that comprisesconversion of pyruvate to acetyl-CoA and formate by a pyruvate formatelyase and an engineered metabolic pathway that comprises conversion ofacetyl-CoA to ethanol by a bifunctional acetaldehyde/alcoholdehydrogenase, further comprising a native and/or heterologous gpd1polynucleotide operably linked to a native gpd2 promoter polynucleotideand a deletion of one or more native enzymes encoded by both an fdh1polynucleotide and an fdh2 polynucleotide.

In some embodiments, the deletion of one or more native enzymes thatfunction to produce glycerol and/or regulate glycerol synthesis in therecombinant microorganism reduces glycerol formation by: more than about10% of the glycerol produced by a recombinant microorganism without adeletion of one or more native enzymes that function to produce glyceroland/or regulate glycerol synthesis; more than about 20% of the glycerolproduced by a recombinant microorganism without a deletion of one ormore native enzymes that function to produce glycerol and/or regulateglycerol synthesis; more than about 30% of the glycerol produced by arecombinant microorganism without a deletion of one or more nativeenzymes that function to produce glycerol and/or regulate glycerolsynthesis; more than about 40% of the glycerol produced by a recombinantmicroorganism without a deletion of one or more native enzymes thatfunction to produce glycerol and/or regulate glycerol synthesis; morethan about 50% of the glycerol produced by a recombinant microorganismwithout a deletion of one or more native enzymes that function toproduce glycerol and/or regulate glycerol synthesis; more than about 60%of the glycerol produced by a recombinant microorganism without adeletion of one or more native enzymes that function to produce glyceroland/or regulate glycerol synthesis; more than about 70% of the glycerolproduced by a recombinant microorganism without a deletion of one ormore native enzymes that function to produce glycerol and/or regulateglycerol synthesis; more than about 80% of the glycerol produced by arecombinant microorganism without a deletion of one or more nativeenzymes that function to produce glycerol and/or regulate glycerolsynthesis; more than about 90% of the glycerol produced by a recombinantmicroorganism without a deletion of one or more native enzymes thatfunction to produce glycerol and/or regulate glycerol synthesis; morethan about 95% of the glycerol produced by a recombinant microorganismwithout a deletion of one or more native enzymes that function toproduce glycerol and/or regulate glycerol synthesis; or more than about99% of the glycerol produced by a recombinant microorganism without adeletion of one or more native enzymes that function to produce glyceroland/or regulate glycerol synthesis.

In some embodiments, the recombinant microorganism produces an amount offormate selected from: at least about 0.012 g/L in 24 hours; at leastabout 0.022 g/L in 48 hours; or at least about 2.5 g/L in 142 hours.

In some embodiments, the recombinant microorganism produces a formateyield selected from: at least about 0.05-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 0.1-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 0.5-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 1.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 5.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 10.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 20.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 30.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 40.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 50.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 75.0-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; or at least about 100-fold more formate than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes.

In some embodiments, the recombinant microorganism produces an ethanolyield selected from: at least about 1% more ethanol than is produced bya recombinant microorganism without activation, upregulation, ordownregulation of one or more native and/or heterologous enzymes; atleast about 2% more ethanol than is produced by a recombinantmicroorganism without activation, upregulation, or downregulation of oneor more native and/or heterologous enzymes; at least about 3% moreethanol than is produced by a recombinant microorganism withoutactivation, upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 4% more ethanol than is produced bya recombinant microorganism without activation, upregulation, ordownregulation of one or more native and/or heterologous enzymes; atleast about 5% more ethanol than is produced by a recombinantmicroorganism without activation, upregulation, or downregulation of oneor more native and/or heterologous enzymes; at least about 10% moreethanol than is produced by a recombinant microorganism withoutactivation, upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 20% more ethanol than is producedby a recombinant microorganism without activation, upregulation, ordownregulation of one or more native and/or heterologous enzymes; atleast about 30% more ethanol than is produced by a recombinantmicroorganism without activation, upregulation, or downregulation of oneor more native and/or heterologous enzymes; at least about 40% moreethanol than is produced by a recombinant microorganism withoutactivation, upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 50% more ethanol than is producedby a recombinant microorganism without activation, upregulation, ordownregulation of one or more native and/or heterologous enzymes; atleast about 60% more ethanol than is produced by a recombinantmicroorganism without activation, upregulation, or downregulation of oneor more native and/or heterologous enzymes; at least about 70% moreethanol than is produced by a recombinant microorganism withoutactivation, upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 80% more ethanol than is producedby a recombinant microorganism without activation, upregulation, ordownregulation of one or more native and/or heterologous enzymes; atleast about 90% more ethanol than is produced by a recombinantmicroorganism without activation, upregulation, or downregulation of oneor more native and/or heterologous enzymes; at least about 95% moreethanol than is produced by a recombinant microorganism withoutactivation, upregulation, or downregulation of one or more native and/orheterologous enzymes; or at least about 99% more ethanol than isproduced by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes.

In some embodiments, the conversion of the carbohydrate source toethanol by the recombinant microorganism, or the enzymes engineeredtherein, is under anaerobic conditions.

In some embodiments, the recombinant microorganism has an acetate uptake(g/L) under anaerobic conditions selected from: at least about 1% moreacetate uptake than that taken up by a recombinant microorganism withoutactivation, upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 10% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 20% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 30% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 40% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 50% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 60% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 70% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 80% more acetate uptake than thattaken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; and at least about 90% more acetate uptake thanthat taken up by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes.

In some embodiments, the recombinant microorganism produces more ethanolat a slower glucose utilization rate compared to a recombinantmicroorganism without deletion of one or more native enzymes thatfunction to produce glycerol and/or regulate glycerol synthesis, whereinthe glucose utilization rate is selected from: at least about 1% lessglucose used per hour than that used by a recombinant microorganismwithout activation, upregulation, or downregulation of one or morenative and/or heterologous enzymes; at least about 5% less glucose usedper hour than that used by a recombinant microorganism withoutactivation, upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 10% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 20% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 30% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 40% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 50% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 60% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 70% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; at least about 80% less glucose used per hour thanthat used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes; and at least about 90% less glucose used per hourthan that used by a recombinant microorganism without activation,upregulation, or downregulation of one or more native and/orheterologous enzymes.

Another aspect of the invention relates to a recombinant microorganismcomprising: one or more heterologous enzymes that function to regulateglycerol synthesis, wherein said one or more heterologous enzymes isactivated, upregulated or downregulated; and one or more native and/orheterologous enzymes that function in one or more engineered metabolicpathways to convert a carbohydrate source to ethanol, wherein said oneor more native and/or heterologous enzymes is activated, upregulated ordownregulated. In certain embodiments, the one or more heterologousenzymes that function to regulate glycerol synthesis are encoded by anfps1 polynucleotide. In one embodiment, the fps1 polynucleotide is fromEscherichia coli.

In some embodiments, one of the engineered metabolic pathways of theabove recombinant microorganism comprises conversion of pyruvate toacetyl-CoA and formate. In certain embodiments, pyruvate is converted toacetyl-CoA and formate by a pyruvate formate lyase (PFL). In someembodiments, PFL is of prokaryotic or eukaryotic origin. In someembodiments, PFL is from one or more of a Bifidobacteria, anEscherichia, a Thermoanaerobacter, a Clostridia, a Streptococcus, aLactobacillus, a Chlamydomonas, a Piromyces, a Neocallimastix, or aBacillus species. In some embodiments, the PFL is from one or more of aBacillus licheniformis, a Streptococcus thermophilus, a Lactobacillusplantarum, a Lactobacillus casei, a Bifidobacterium adolescentis, aClostridium cellulolyticum, a Escherichia coli, a Chlamydomonasreinhardtii PflA, a Piromyces sp. E2, or a Neocallimastix frontalis. Inone embodiment, PFL is from a Bifidobacterium adolescentis.

In some embodiments, one of said engineered metabolic pathways of theabove recombinant microorganism comprises conversion of acetyl-CoA toethanol. In some embodiments, acetyl-CoA is converted to acetaldehyde byan acetaldehyde dehydrogenase and acetaldehyde is converted to ethanolby an alcohol dehydrogenase. In other embodiments, acetyl-CoA isconverted to ethanol by a bifunctional acetaldehyde/alcoholdehydrogenase. In some embodiments, the acetaldehyde dehydrogenase,alcohol dehydrogenase, or bifuntional acetaldehyde/alcohol dehydrogenaseis of prokaryotic or eukaryotic origin. In one embodiment, acetaldehydedehydrogenase is from C. phytofermentans. In certain embodiments, thebifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia,a Clostridia, a Chlamydomonas, a Piromyces, or a Bifidobacteria species.In some embodiments, the bifunctional acetaldehyde/alcohol dehydrogenaseis from Escherichia coli, Clostridium phytofermentans, Chlamydomonasreinhardtii, Piromyces sp. E2, or Bifidobacterium adolescentis. In oneembodiment, the bifunctional acetaldehyde/alcohol dehydrogenase is froma Bifidobacterium adolescentis or Piromyces sp. E2.

In further aspects, the recombinant microorganism comprises a deletionof one or more native enzymes encoded by an fdh1 polynucleotide, an fdh2polynucleotide, or both an fdh1 polynucleotide and an fdh2polynucleotide.

In some embodiments, the recombinant microorganism produces ethanol. Inother embodiments, the recombinant microorganism produces formate. Insome embodiments, the recombinant microorganism is selected from thegroup consisting of Saccharomyces cerevisiae, Kluyveromyces lactis,Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenulapolymorphs, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans,Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus,Schizosaccharomyces pombe, Candida albicans, and Schwanniomycesoccidentalis. In one embodiment, the recombinant microorganism isSaccharomyces cerevisiae.

In some embodiments, the recombinant microorganisms of the inventionfurther comprise one or more native and/or heterologous enzymes thatfunction in one or more engineered metabolic pathways to convert xyloseto xylulose-5-phosphate and/or arabinose to xylulose-5-phosphate,wherein the one or more native and/or heterologous enzymes areactivated, upregulated or downregulated.

In some embodiments, the recombinant microorganisms of the inventionfurther comprise one or more native and/or heterologous enzymes whichencodes a saccharolytic enzyme, including amylases, cellulases,hemicellulases, cellulolytic and amylolytic accessory enzymes,inulinases, levanases, and pentose sugar utilizing enzymes. In oneaspect, the saccharolytic enzyme is an amylase, where the amylase isselected from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C.lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M.darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus,Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum,Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagusdegradans, Piromyces equii, Neocallimastix patricarum or Arabidopsisthaliana. In another aspect, the saccharolytic enzyme is an amylase fromS. fibuligera glucoamylase (glu-0111-00).

Another aspect of the invention relates to a method for decreasingcellular glycerol comprising contacting biomass with a recombinantmicroorganism of the invention. A further aspect of the inventionrelates to a method for increasing cytosolic formate comprisingcontacting biomass with a recombinant microorganism of the invention.Another aspect of the invention relates to a process for convertingbiomass to ethanol comprising contacting biomass with a recombinantmicroorganism of the invention. In some embodiments, the biomasscomprises lignocellulosic biomass. In some embodiments, thelignocellulosic biomass is selected from the group consisting of grass,switch grass, cord grass, rye grass, reed canary grass, mixed prairiegrass, miscanthus, sugar-processing residues, sugarcane bagasse,sugarcane straw, agricultural wastes, rice straw, rice hulls, barleystraw, corn cobs, cereal straw, wheat straw, canola straw, oat straw,oat hulls, corn fiber, stover, soybean stover, corn stover, forestrywastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood,softwood, agave, and combinations thereof. In some embodiments, thebiomass is corn mash or corn starch.

In another aspect, the present invention also describes industrial yeaststrains that express enzymes for the production of fuel ethanol fromcorn starch.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows a schematic of high yield metabolism.

FIG. 2 depicts the glycolysis pathway.

FIG. 3 shows a schematic of the glycolysis/fermentation pathway.

FIG. 4 shows a map depicting location of primers used to make markeddeletion of GPD1.

FIG. 5 shows a map depicting location of primers used to remove markerfrom GPD1 locus.

FIG. 6 shows a map depicting location of primers used to make markeddeletion of GPD2.

FIG. 7 shows a map depicting location of primers used to remove markerfrom GPD2 locus.

FIG. 8 shows a map depicting location of primers used to make markeddeletion of FDH1.

FIG. 9 shows a map depicting location of primers used to remove markerfrom FDH1 locus.

FIG. 10 shows a map depicting location of primers used to make markeddeletion of FDH2.

FIG. 11 shows a map depicting location of primers used to remove markerfrom FDH2 locus.

FIG. 12 shows an alignment of PFL enzymes from various organisms.

FIG. 13 shows a graph of formate production over 48 hours.

FIG. 14 shows a graph of formate production at the end of 142 hours inmicroaerobic and anaerobic conditions.

FIG. 15 shows a graph of the growth of strains of the invention over 72hours as measured by OD.

FIG. 16 shows a graph of glycerol production (g/L) of strains of theinvention over 72 hours.

FIG. 17 shows a graph of ethanol production (g/L) of strains of theinvention over 72 hours.

FIG. 18 shows a graph of glucose utilization (g/L) of strains of theinvention over 72 hours.

FIG. 19 shows a graph of the growth of a strain of the invention over142 hours as measured by OD.

FIG. 20 shows a graph of the relative growth rate (mOD/min) of strainsof the invention.

FIG. 21 shows a graph of ethanol production (g/L) of strains of theinvention after 50 hours of fermentation.

FIG. 22 shows a graph of ethanol production (g/L) of strains of theinvention after 50 hours of fermentation.

FIG. 23 shows a graph of glycerol production (g/L) of strains of theinvention after 50 hours of fermentation.

FIG. 24 shows a graph of glycerol production (g/L) of strains of theinvention after 50 hours of fermentation.

FIG. 25 shows a graph of glucose utilization (g/L), glycerol production(g/L), and ethanol production (g/L) after 72 hours of fermentation.

FIG. 26 shows a diagram depicting integration of E. coli AADHs at theGPD1 locus.

FIG. 27 shows a diagram depicting integration of E. coli AADHs at theFCY1 locus

FIG. 28 shows a schematic diagram of a strategy for PCR construction andintegration of KT-MX and NT-MX integration cassettes into bothchromosomes of a target loci.

FIG. 29 shows a schematic diagram of strategy used to replace integratedKT-MX and NT-MX selection cassettes with a Mascoma Assembly on bothchromosomes of a target loci.

FIG. 30 shows a molecular map and genotyping of MA0370 integrated at theFDH1 site of M3625.

FIG. 31 shows an image of an agarose gel containing PCR products used togenotype and sequence the MA0370 site.

FIG. 32 shows a molecular map and genotyping of MA0280 integrated at theFDH2 site of M3625.

FIG. 33 shows an image of an agarose gel containing PCR products used togenotype and sequence the MA0280 site.

FIG. 34 shows a molecular map and genotyping of MA0289 integrated at theGPD2 site of M3625.

FIG. 35 shows an image of an agarose gel containing PCR products used togenotype and sequence the MA0370 site.

FIG. 36 shows a molecular map and genotyping of MA0317 integrated at theFCY1 site of M3625.

FIG. 37 shows an image of an agarose gel containing PCR products used togenotype and sequence the MA0317 site.

FIG. 38 shows a graph depicting the results of a starch assay performedwith strains M2390, M2519, M2691, M3498, and M3625.

FIG. 39 shows an anti-peptide Western blot analysis of cell extracts(PflA, PflB, AdhE) and aerobic culture supernatants (AE9).

FIG. 40 shows a graph depicting the results of a formate lyase assayperformed with engineered strains M3465, M3625, M3679, M3680, and M2390.

FIG. 41 shows a graph depicting the results of an alcohol dehydrogenaseassay performed with engineered strains M3465, M3625, M3679, and M3680.

FIG. 42 shows a graph depicting the results of a glucoamylase activityassay performed with engineered strains M3625 and M3680 using 50 μg/mLAE9 on corn starch at room temperature (˜25° C.).

FIG. 43A is a schematic showing insertion of promoters and terminatorsused to express, GPD2, and B. adolescentis pflA, pflB and adhE at theGPD1 locus in M3624 and M3515.

FIG. 43B is a schematic showing insertion of promoters and terminatorsused to express GPD1 and B. adolescentis pflA, pflB and adhE at the GPD2locus in M3624 and M3515.

FIG. 43C is a schematic showing deletion of the FDH1 gene in M3624 andM3515.

FIG. 43D is a schematic showing insertions of promoters and terminatorsused to express B. adolescentis pflA, pflB and adhE at the FDH2 locus inM3624 and M3515.

FIG. 44A shows HPLC analysis of formate produced by glycerol reductionstrains during fermentation of 28% solids corn mash.

FIG. 44B shows HPLC analysis of glycerol produced by glycerol reductionstrains during fermentation of 28% solids corn mash.

FIG. 44C shows HPLC analysis of ethanol produced by glycerol reductionstrains during fermentation of 28% solids corn mash.

FIG. 45A is a schematic showing insertion of promoters and terminatorsused to express B. adolescentis pflA, pflB and adhE at the GPD2 locus inM3465.

FIG. 45B is a schematic showing deletion of the FDH1 gene in M3465.

FIG. 45C is a schematic showing insertion of promoters and terminatorsused to express B. adolescentis pflA, pflB and adhE at the FDH2 locus inM3465.

FIG. 46A is a schematic showing insertion of promoters and terminatorsused to express B. adolescentis pflA, pflB and adhE at the GPD1 locus inM3469.

FIG. 46B is a schematic showing deletion of the FDH1 gene in M3469.

FIG. 46C is a schematic showing insertion of promoters and terminatorsused to express B. adolescentis pflA, pflB and adhE at the FDH2 locus inM3469.

FIG. 47 shows HPLC analysis of ethanol titers produced by glycerolreduction strains during fermentation of 30% solids corn mash.

FIG. 48 shows HPLC analysis of glycerol titers produced by glycerolreduction strains during fermentation of 30% solids corn mash.

FIG. 49 shows the reverse reaction catalyzed by Bifidobacteriumadolecentis bifunctional alcohol dehydrogenase (AdhE) in which ethanolis converted to acetaldehyde.

FIG. 50 shows a diagram of the reaction for the conversion ofacetaldehyde to acetyl CoA by AdhE.

FIG. 51 is a schematic showing insertion of promoters and terminatorsused to express B. adolescentis pflA, pflB and adhE at the GPP1 locus inTB655.

FIG. 52 is a schematic showing insertion of promoters and terminatorsused to express B. adolescentis pflA, pflB and adhE at the GPP2 locus inTB656.

FIG. 53 is a graph depicting decreased glycerol formation in strainsTB655 and TB656 compared to strain M3297.

FIG. 54 is a graph depicting increased ethanol yield in strains TB655and TB656 compared to strain M3297.

FIG. 55 is a graph depicting formate production in strains TB655, TB656,and M3297.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “heterologous” when used in reference to a polynucleotide, agene, a polypeptide, or an enzyme refers to a polynucleotide, gene,polypeptide, or an enzyme not normally found in the host organism.“Heterologous” also includes a native coding region, or portion thereof,that is reintroduced into the source organism in a form that isdifferent from the corresponding native gene, e.g., not in its naturallocation in the organism's genome. The heterologous polynucleotide orgene may be introduced into the host organism by, e.g., gene transfer. Aheterologous gene may include a native coding region that is a portionof a chimeric gene including non-native regulatory regions that isreintroduced into the native host. Foreign genes can comprise nativegenes inserted into a non-native organism, or chimeric genes.

The term “heterologous polynucleotide” is intended to include apolynucleotide that encodes one or more polypeptides or portions orfragments of polypeptides. A heterologous polynucleotide may be derivedfrom any source, e.g., eukaryotes, prokaryotes, viruses, or syntheticpolynucleotide fragments.

The terms “promoter” or “surrogate promoter” is intended to include apolynucleotide that can transcriptionally control a gene-of-interestthat it does not transcriptionally control in nature. In certainembodiments, the transcriptional control of a surrogate promoter resultsin an increase in expression of the gene-of-interest. In certainembodiments, a surrogate promoter is placed 5′ to the gene-of-interest.A surrogate promoter may be used to replace the natural promoter, or maybe used in addition to the natural promoter. A surrogate promoter may beendogenous with regard to the host cell in which it is used, or it maybe a heterologous polynucleotide sequence introduced into the host cell,e.g., exogenous with regard to the host cell in which it is used.

The terms “gene(s)” or “polynucleotide” or “polynucleotide sequence(s)”are intended to include nucleic acid molecules, e.g., polynucleotideswhich include an open reading frame encoding a polypeptide, and canfurther include non-coding regulatory sequences, and introns. Inaddition, the terms are intended to include one or more genes that mapto a functional locus. In addition, the terms are intended to include aspecific gene for a selected purpose. The gene may be endogenous to thehost cell or may be recombinantly introduced into the host cell, e.g.,as a plasmid maintained episomally or a plasmid (or fragment thereof)that is stably integrated into the genome. In addition to the plasmidform, a gene may, for example, be in the form of linear DNA. In certainembodiments, the gene or polynucleotide is involved in at least one stepin the bioconversion of biomass to, e.g., ethanol. Accordingly, the termis intended to include any gene encoding a polypeptide, such as theenzymes acetate kinase (ACK), phosphotransacetylase (PTA), lactatedehydrogenase (LDH), pyruvate formate lyase (PFL), aldehydedehydrogenase (ADH) and/or alcohol dehydrogenase (ADH), acetyl-CoAtransferase (ACS), acetaldehyde dehydrogenase (ACDH),acetaldehyde/alcohol dehydrogenase (AADH), glycerol-3-phosphatedehydrogenase (GPD), glycerol 3-phosphatase (GPP), acetyl-CoAsynthetase, thiolase, CoA transferase, acetoacetate decarboxylase,alcohol acetyltransferase enzymes in the D-xylose pathway, such asxylose isomerase and xylulokinase, enzymes in the L-arabinose pathway,such as L-arabinose isomerase and L-ribulose-5-phosphate 4-epimerase.The term gene is also intended to cover all copies of a particular gene,e.g., all of the DNA sequences in a cell encoding a particular geneproduct.

The term “transcriptional control” is intended to include the ability tomodulate gene expression at the level of transcription. In certainembodiments, transcription, and thus gene expression, is modulated byreplacing or adding a surrogate promoter near the 5′ end of the codingregion of a gene-of-interest, thereby resulting in altered geneexpression. In certain embodiments, the transcriptional control of oneor more genes is engineered to result in the optimal expression of suchgenes, e.g., in a desired ratio. The term also includes inducibletranscriptional control as recognized in the art.

The term “expression” is intended to include the expression of a gene atleast at the level of mRNA production.

The term “expression product” is intended to include the resultantproduct, e.g., a polypeptide, of an expressed gene.

The term “increased expression” is intended to include an alteration ingene expression at least at the level of increased mRNA production and,preferably, at the level of polypeptide expression. The term “increasedproduction” is intended to include an increase in the amount of apolypeptide expressed, in the level of the enzymatic activity of thepolypeptide, or a combination thereof, as compared to the nativeproduction of, or the enzymatic activity, of the polypeptide.

The terms “activity,” “activities,” “enzymatic activity,” and “enzymaticactivities” are used interchangeably and are intended to include anyfunctional activity normally attributed to a selected polypeptide whenproduced under favorable conditions. Typically, the activity of aselected polypeptide encompasses the total enzymatic activity associatedwith the produced polypeptide. The polypeptide produced by a host celland having enzymatic activity may be located in the intracellular spaceof the cell, cell-associated, secreted into the extracellular milieu, ora combination thereof. Techniques for determining total activity ascompared to secreted activity are described herein and are known in theart.

The term “xylanolytic activity” is intended to include the ability tohydrolyze glycosidic linkages in oligopentoses and polypentoses.

The term “arabinolytic activity” is intended to include the ability tohydrolyze glycosidic linkages in oligopentoses and polypentoses.

The term “cellulolytic activity” is intended to include the ability tohydrolyze glycosidic linkages in oligohexoses and polyhexoses.Cellulolytic activity may also include the ability to depolymerize ordebranch cellulose and hemicellulose.

As used herein, the term “lactate dehydrogenase” or “LDH” is intended toinclude the enzymes capable of converting pyruvate into lactate. It isunderstood that LDH can also catalyze the oxidation of hydroxybutyrate.LDH includes those enzymes that correspond to Enzyme Commission Number1.1.1.27.

As used herein the term “alcohol dehydrogenase” or “ADH” is intended toinclude the enzymes capable of converting acetaldehyde into an alcohol,such as ethanol. ADH also includes the enzymes capable of convertingacetone to isopropanol. ADH includes those enzymes that correspond toEnzyme Commission Number 1.1.1.1.

As used herein, the term “phosphotransacetylase” or “PTA” is intended toinclude the enzymes capable of converting acetyl-phosphate intoacetyl-CoA. PTA includes those enzymes that correspond to EnzymeCommission Number 2.3.1.8.

As used herein, the term “acetate kinase” or “ACK” is intended toinclude the enzymes capable of converting acetate into acetyl-phosphate.ACK includes those enzymes that correspond to Enzyme Commission Number2.7.2.1.

As used herein, the term “pyruvate formate lyase” or “PFL” is intendedto include the enzymes capable of converting pyruvate into acetyl-CoAand formate. PFL includes those enzymes that correspond to EnzymeCommission Number 2.3.1.54.

As used herein, the term “formate dehydrogenase” or “FDH” is intended toinclude the enzymes capable of converting formate and NAD⁺ to NADH andCO₂. FDH includes those enzymes that correspond to Enzyme CommissionNumber 1.2.1.2.

As used herein, the term “acetaldehyde dehydrogenase” or “ACDH” isintended to include the enzymes capable of converting acetyl-CoA toacetaldehyde. ACDH includes those enzymes that correspond to EnzymeCommission Number 1.2.1.3.

As used herein, the term “acetaldehyde/alcohol dehydrogenase” isintended to include the enzymes capable of converting acetyl-CoA toethanol. Acetaldehyde/alcohol dehydrogenase includes those enzymes thatcorrespond to Enzyme Commission Numbers 1.2.1.10 and 1.1.1.1.

As used herein, the term “glycerol-3-phosphate dehydrogenase” or “GPD”is intended to include the enzymes capable of convertingdihydroxyacetone phosphate to glycerol-3-phosphate. GPD includes thoseenzymes that correspond to Enzyme Commission Number 1.1.1.8.

As used herein, the term “glycerol 3-phosphatase” or “GPP” is intendedto include the enzymes capable of converting glycerol 3-phosphate toglycerol. GPP includes those enzymes that correspond to EnzymeCommission Number 3.1.3.21.

As used herein, the term “acetyl-CoA synthetase” or “ACS” is intended toinclude the enzymes capable of converting acetate to acetyl-CoA.Acetyl-CoA synthetase includes those enzymes that correspond to EnzymeCommission Number 6.2.1.1.

As used herein, the term “thiolase” is intended to include the enzymescapable of converting acetyl-CoA to acetoacetyl-CoA. Thiolase includesthose enzymes that correspond to Enzyme Commission Number 2.3.1.9.

As used herein, the term “CoA transferase” is intended to include theenzymes capable of converting acetate and acetoacetyl-CoA toacetoacetate and acetyl-CoA. CoA transferase includes those enzymes thatcorrespond to Enzyme Commission Number 2.8.3.8.

As used herein, the term “acetoacetate decarboxylase” is intended toinclude the enzymes capable of converting acetoacetate to acetone andcarbon dioxide. Acetoacetate decarboxylase includes those enzymes thatcorrespond to Enzyme Commission Number 4.1.1.4.

As used herein, the term “alcohol acetyltransferase” is intended toinclude the enzymes capable of converting acetyl-CoA and ethanol toethyl acetate. Alcohol acetyltransferase includes those enzymes thatcorrespond to Enzyme Commission Number 2.3.1.84.

The term “pyruvate decarboxylase activity” is intended to include theability of a polypeptide to enzymatically convert pyruvate intoacetaldehyde and carbon dioxide (e.g., “pyruvate decarboxylase” or“PDC”). Typically, the activity of a selected polypeptide encompassesthe total enzymatic activity associated with the produced polypeptide,comprising, e.g., the superior substrate affinity of the enzyme,thermostability, stability at different pHs, or a combination of theseattributes. PDC includes those enzymes that correspond to EnzymeCommission Number 4.1.1.1.

The term “ethanologenic” is intended to include the ability of amicroorganism to produce ethanol from a carbohydrate as a fermentationproduct. The term is intended to include, but is not limited to,naturally occurring ethanologenic organisms, ethanologenic organismswith naturally occurring or induced mutations, and ethanologenicorganisms which have been genetically modified.

The terms “fermenting” and “fermentation” are intended to include theenzymatic process (e.g., cellular or acellular, e.g., a lysate orpurified polypeptide mixture) by which ethanol is produced from acarbohydrate, in particular, as a product of fermentation.

The term “secreted” is intended to include the movement of polypeptidesto the periplasmic space or extracellular milieu. The term “increasedsecretion” is intended to include situations in which a givenpolypeptide is secreted at an increased level (i.e., in excess of thenaturally-occurring amount of secretion). In certain embodiments, theterm “increased secretion” refers to an increase in secretion of a givenpolypeptide that is at least about 10% or at least about 100%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared tothe naturally-occurring level of secretion.

The term “secretory polypeptide” is intended to include anypolypeptide(s), alone or in combination with other polypeptides, thatfacilitate the transport of another polypeptide from the intracellularspace of a cell to the extracellular milieu. In certain embodiments, thesecretory polypeptide(s) encompass all the necessary secretorypolypeptides sufficient to impart secretory activity to a Gram-negativeor Gram-positive host cell or to a yeast host cell. Typically, secretoryproteins are encoded in a single region or locus that may be isolatedfrom one host cell and transferred to another host cell using geneticengineering. In certain embodiments, the secretory polypeptide(s) arederived from any bacterial cell having secretory activity or any yeastcell having secretory activity. In certain embodiments, the secretorypolypeptide(s) are derived from a host cell having Type II secretoryactivity. In certain embodiments, the host cell is a thermophilicbacterial cell. In certain embodiments, the host cell is a yeast cell.

The term “derived from” is intended to include the isolation (in wholeor in part) of a polynucleotide segment from an indicated source or thepurification of a polypeptide from an indicated source. The term isintended to include, for example, direct cloning, PCR amplification, orartificial synthesis from or based on a sequence associated with theindicated polynucleotide source.

The term “recombinant microorganism” or “recombinant host cell” isintended to include progeny or derivatives of the recombinantmicroorganisms of the invention. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny or derivatives may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

By “thermophilic” is meant an organism that thrives at a temperature ofabout 45° C. or higher.

By “mesophilic” is meant an organism that thrives at a temperature ofabout 20-45° C.

The term “organic acid” is art-recognized. “Organic acid,” as usedherein, also includes certain organic solvents such as ethanol. The term“lactic acid” refers to the organic acid 2-hydroxypropionic acid ineither the free acid or salt form. The salt form of lactic acid isreferred to as “lactate” regardless of the neutralizing agent, i.e.,calcium carbonate or ammonium hydroxide. The term “acetic acid” refersto the organic acid methanecarboxylic acid, also known as ethanoic acid,in either free acid or salt form. The salt form of acetic acid isreferred to as “acetate.”

Certain embodiments of the present invention provide for the“insertion,” (e.g., the addition, integration, incorporation, orintroduction) of certain genes or particular polynucleotide sequenceswithin thermophilic or mesophilic microorganisms, which insertion ofgenes or particular polynucleotide sequences may be understood toencompass “genetic modification(s)” or “transformation(s)” such that theresulting strains of said thermophilic or mesophilic microorganisms maybe understood to be “genetically modified” or “transformed.” In certainembodiments, strains may be of bacterial, fungal, or yeast origin.

Certain embodiments of the present invention provide for the“inactivation” or “deletion” of certain genes or particularpolynucleotide sequences within thermophilic or mesophilicmicroorganisms, which “inactivation” or “deletion” of genes orparticular polynucleotide sequences may be understood to encompass“genetic modification(s)” or “transformation(s)” such that the resultingstrains of said thermophilic or mesophilic microorganisms may beunderstood to be “genetically modified” or “transformed.” In certainembodiments, strains may be of bacterial, fungal, or yeast origin.

The term “consolidated bioprocessing” or “CBP” refers to biomassprocessing schemes involving enzymatic or microbial hydrolysis thatcommonly involve four biologically mediated transformations: (1) theproduction of saccharolytic enzymes (amylases, cellulases, andhemicellulases); (2) the hydrolysis of carbohydrate components presentin pretreated biomass to sugars; (3) the fermentation of hexose sugars(e.g., glucose, mannose, and galactose); and (4) the fermentation ofpentose sugars (e.g., xylose and arabinose). These four transformationsoccur in a single step in a process configuration called CBP, which isdistinguished from other less highly integrated configurations in thatit does not involve a dedicated process step for cellulase and/orhemicellulase production.

The term “CBP organism” is intended to include microorganisms of theinvention, e.g., microorganisms that have properties suitable for CBP.

In one aspect of the invention, the genes or particular polynucleotidesequences are inserted to activate the activity for which they encode,such as the expression of an enzyme. In certain embodiments, genesencoding enzymes in the metabolic production of ethanol, e.g., enzymesthat metabolize pentose and/or hexose sugars, may be added to amesophilic or thermophilic organism. In certain embodiments of theinvention, the enzyme may confer the ability to metabolize a pentosesugar and be involved, for example, in the D-xylose pathway and/orL-arabinose pathway.

In one aspect of the invention, the genes or particular polynucleotidesequences are partially, substantially, or completely deleted, silenced,inactivated, or down-regulated in order to inactivate the activity forwhich they encode, such as the expression of an enzyme. Deletionsprovide maximum stability because there is no opportunity for a reversemutation to restore function. Alternatively, genes can be partially,substantially, or completely deleted, silenced, inactivated, ordown-regulated by insertion of nucleic acid sequences that disrupt thefunction and/or expression of the gene (e.g., P1 transduction or othermethods known in the art). The terms “eliminate,” “elimination,” and“knockout” are used interchangeably with the terms “deletion,” “partialdeletion,” “substantial deletion,” or “complete deletion.” In certainembodiments, strains of thermophilic or mesophilic microorganisms ofinterest may be engineered by site directed homologous recombination toknockout the production of organic acids. In still other embodiments,RNAi or antisense DNA (asDNA) may be used to partially, substantially,or completely silence, inactivate, or down-regulate a particular gene ofinterest.

In certain embodiments, the genes targeted for deletion or inactivationas described herein may be endogenous to the native strain of themicroorganism, and may thus be understood to be referred to as “nativegene(s)” or “endogenous gene(s).” An organism is in “a native state” ifit has not been genetically engineered or otherwise manipulated by thehand of man in a manner that intentionally alters the genetic and/orphenotypic constitution of the organism. For example, wild-typeorganisms may be considered to be in a native state. In otherembodiments, the gene(s) targeted for deletion or inactivation may benon-native to the organism.

Similarly, the enzymes of the invention as described herein can beendogenous to the native strain of the microorganism, and can thus beunderstood to be referred to as “native” or “endogenous.”

The term “upregulated” means increased in activity, e.g., increase inenzymatic activity of the enzyme as compared to activity in a nativehost organism.

The term “downregulated” means decreased in activity, e.g., decrease inenzymatic activity of the enzyme as compared to activity in a nativehost organism.

The term “activated” means expressed or metabolically functional.

The term “adapted for growing” means selection of an organism for growthunder conditions in which the organism does not otherwise grow or inwhich the organism grows slowly or minimally. Thus, an organism that issaid to be adapted for growing under the selected condition, growsbetter than an organism that has not been adapted for growing under theselected conditions. Growth can be measured by any methods known in theart, including, but not limited to, measurement of optical density orspecific growth rate.

The term “carbohydrate source” is intended to include any source ofcarbohydrate including, but not limited to, biomass or carbohydrates,such as a sugar or a sugar alcohol. “Carbohydrates” include, but are notlimited to, monosaccharides (e.g., glucose, fructose, galactose, xylose,arabinose, or ribose), sugar derivatives (e.g., sorbitol, glycerol,galacturonic acid, rhamnose, xylitol), disaccharides (e.g., sucrose,cellobiose, maltose, or lactose), oligosaccharides (e.g., xylooligomers,cellodextrins, or maltodextrins), and polysaccharides (e.g., xylan,cellulose, starch, mannan, alginate, or pectin).

As used herein, an “amylolytic enzyme” can be any enzyme involved inamylase digestion, metabolism and/or hydrolysis. The term “amylase”refers to an enzyme that breaks starch down into sugar. Amylase ispresent in human saliva, where it begins the chemical process ofdigestion. Foods that contain much starch but little sugar, such as riceand potato, taste slightly sweet as they are chewed because amylaseturns some of their starch into sugar in the mouth. The pancreas alsomakes amylase (α-amylase) to hydrolyse dietary starch into disaccharidesand trisaccharides which are converted by other enzymes to glucose tosupply the body with energy. Plants and some bacteria also produceamylase. All amylases are glycoside hydrolases and act onα-1,4-glycosidic bonds. Some amylases, such as γ-amylase (glucoamylase),also act on α-1,6-glycosidic bonds. Amylase enzymes include α-amylase(EC 3.2.1.1), β-amylase (EC 3.2.1.2), and γ-amylase (EC 3.2.1.3). Theα-amylases are calcium metalloenzymes, unable to function in the absenceof calcium. By acting at random locations along the starch chain,α-amylase breaks down long-chain carbohydrates, ultimately yieldingmaltotriose and maltose from amylose, or maltose, glucose and “limitdextrin” from amylopectin. Because it can act anywhere on the substrate,α-amylase tends to be faster-acting than β-amylase. In animals, it is amajor digestive enzyme and its optimum pH is about 6.7-7.0. Another formof amylase, β-amylase is also synthesized by bacteria, fungi, andplants. Working from the non-reducing end, β-amylase catalyzes thehydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucoseunits (maltose) at a time. Many microbes produce amylase to degradeextracellular starches. In addition to cleaving the lastα(1-4)glycosidic linkages at the nonreducing end of amylose andamylopectin, yielding glucose, γ-amylase will cleave α(1-6) glycosidiclinkages. Another amylolytic enzyme is alpha-glucosidase that acts onmaltose and other short malto-oligosaccharides produced by alpha-,beta-, and gamma-amylases, converting them to glucose. Anotheramylolytic enzyme is pullulanase. Pullulanase is a specific kind ofglucanase, an amylolytic exoenzyme, that degrades pullulan. Pullulan isregarded as a chain of maltotriose units linked by alpha-1,6-glycosidicbonds. Pullulanase (EC 3.2.1.41) is also known aspullulan-6-glucanohydrolase (Debranching enzyme). Another amylolyticenzyme, isopullulanase, hydrolyses pullulan to isopanose(6-alpha-maltosylglucose). Isopullulanase (EC 3.2.1.57) is also known aspullulan 4-glucanohydrolase. An “amylase” can be any enzyme involved inamylase digestion, metabolism and/or hydrolysis, including α-amylase,β-amylase, glucoamylase, pullulanase, isopullulanase, andalpha-glucosidase.

As used herein, a “saccharolytic enzyme” can be any enzyme involved incarbohydrate digestion, metabolism and/or hydrolysis, includingamylases, cellulases, hemicellulases, cellulolytic and amylolyticaccessory enzymes, inulinases, levanases, and pentose sugar utilizingenzymes.

Biomass

Biomass can include any type of biomass known in the art or describedherein. For example, biomass can include, but is not limited to, starch,sugar, and lignocellulosic materials. Starch materials can include, butare not limited to, mashes such as corn, wheat, rye, barley, rice, ormilo. Sugar materials can include, but are not limited to, sugar beets,artichoke tubers, sweet sorghum, or cane. The terms “lignocellulosicmaterial,” “lignocellulosic substrate,” and “cellulosic biomass” meanany type of biomass comprising cellulose, hemicellulose, lignin, orcombinations thereof, such as but not limited to woody biomass, foragegrasses, herbaceous energy crops, non-woody-plant biomass, agriculturalwastes and/or agricultural residues, forestry residues and/or forestrywastes, paper-production sludge and/or waste paper sludge,waste-water-treatment sludge, municipal solid waste, corn fiber from wetand dry mill corn ethanol plants, and sugar-processing residues. Theterms “hemicellulosics,” “hemicellulosic portions,” and “hemicellulosicfractions” mean the non-lignin, non-cellulose elements oflignocellulosic material, such as but not limited to hemicellulose(i.e., comprising xyloglucan, xylan, glucuronoxylan, arabinoxylan,mannan, glucomannan, and galactoglucomannan, inter alia), pectins (e.g.,homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan),and proteoglycans (e.g., arabinogalactan-protein, extensin, andproline-rich proteins).

In a non-limiting example, the lignocellulosic material can include, butis not limited to, woody biomass, such as recycled wood pulp fiber,sawdust, hardwood, softwood, and combinations thereof; grasses, such asswitch grass, cord grass, rye grass, reed canary grass, miscanthus, or acombination thereof sugar-processing residues, such as but not limitedto sugar cane bagasse; agricultural wastes, such as but not limited torice straw, rice hulls, barley straw, corn cobs, cereal straw, wheatstraw, canola straw, oat straw, oat hulls, and corn fiber; stover, suchas but not limited to soybean stover, corn stover; succulents, such asbut not limited to, Agave; and forestry wastes, such as but not limitedto, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak,maple, birch, willow), softwood, or any combination thereof.Lignocellulosic material may comprise one species of fiber;alternatively, lignocellulosic material may comprise a mixture of fibersthat originate from different lignocellulosic materials. Otherlignocellulosic materials are agricultural wastes, such as cerealstraws, including wheat straw, barley straw, canola straw and oat straw;corn fiber; stovers, such as corn stover and soybean stover; grasses,such as switch grass, reed canary grass, cord grass, and miscanthus; orcombinations thereof.

Paper sludge is also a viable feedstock for lactate or acetateproduction. Paper sludge is solid residue arising from pulping andpaper-making, and is typically removed from process wastewater in aprimary clarifier. At a disposal cost of $30/wet ton, the cost of sludgedisposal equates to $5/ton of paper that is produced for sale. The costof disposing of wet sludge is a significant incentive to convert thematerial for other uses, such as conversion to ethanol. Processesprovided by the present invention are widely applicable. Moreover, thesaccharification and/or fermentation products may be used to produceethanol or higher value added chemicals, such as organic acids,aromatics, esters, acetone and polymer intermediates.

Glycerol Reduction

Anaerobic growth conditions require the production of endogenouseelectron acceptors, such as the coenzyme nicotinamide adeninedinucleotide (NAD⁺). In cellular redox reactions, the NAD⁺/NADH coupleplays a vital role as a reservoir and carrier of reducing equivalents.Ansell, R., et al., EMBO J. 16:2179-87 (1997). Cellular glycerolproduction, which generates an NAD⁺, serves as a redox valve to removeexcess reducing power during anaerobic fermentation in yeast. Glycerolproduction is, however, an energetically wasteful process that expendsATP and results in the loss of a reduced three-carbon compound. Ansell,R., et al., EMBO J. 16:2179-87 (1997). To generate glycerol from astarting glucose molecule, glycerol 3-phosphate dehydrogenase (GPD)reduces dihydroxyacetone phosphate to glycerol 3-phosphate and glycerol3-phosphatase (GPP) dephosphorylates glycerol 3-phosphate to glycerol.Despite being energetically wasteful, glycerol production is a necessarymetabolic process for anaerobic growth as deleting GPD activitycompletely inhibits growth under anaeroblic conditions. See Ansell, R.,et al., EMBO J. 16:2179-87 (1997).

GPD is encoded by two isogenes, gpd1 and gpd2. GPD1 encodes the majorisoform in anaerobically growing cells, while GPD2 is required forglycerol production in the absence of oxygen, which stimulates itsexpression. Pahlman, A-K., et al., J. Biol. Chem. 276:3555-63 (2001).The first step in the conversion of dihydroxyacetone phosphate toglycerol by GPD is rate controlling. Guo, Z. P., et al., Metab. Eng.13:49-59 (2011). GPP is also encoded by two isogenes, gpp1 and gpp2. Thedeletion of GPP genes arrests growth when shifted to anaerobicconditions, demonstrating that GPP is important for cellular toleranceto osmotic and anaerobic stress. See Pahlman, A-K., et al., J. Biol.Chem. 276:3555-63 (2001).

Because glycerol is a major by-product of anaerobic production ofethanol, many efforts have been made to delete cellular production ofglycerol. However, because of the reducing equivalents produced byglycerol synthesis, deletion of the glycerol synthesis pathway cannot bedone without compensating for this valuable metabolic function. Attemptsto delete glycerol production and engineer alternate electron acceptorshave been made. Lidén, G., et al., Appl. Env. Microbiol. 62:3894-96(1996); Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010).Lidén and Medina both deleted the gpd1 and gpd2 genes and attempted tobypass glycerol formation using additional carbon sources. Lidénengineered a xylose reductase from Pichia stipitis into an S. cerevisiaegpd1/2 deletion strain. The xylose reductase activity facilitated theanaerobic growth of the glycerol-deleted strain in the presence ofxylose. See Lidén, G., et al., Appl. Env. Microbiol. 62:3894-96 (1996).Medina engineered an acetylaldehyde dehydrogenase, mhpF, from E. coliinto an S. cerevisiae gpd1/2 deletion strain to convert acetyl-CoA toacetaldehyde. The acetylaldehyde dehydrogenase activity facilitated theanaerobic growth of the glycerol-deletion strain in the presence ofacetic acid but not in the presence of glucose as the sole source ofcarbon. Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010);see also EP 2277989. Medina noted several issues with themhpF-containing strain that needed to be addressed before implementingindustrially, including significantly reduced growth and productformation rates than yeast comprising GPD1 and GPD2.

Additional attempts to redirect flux from glycerol to ethanol haveincluded the engineering of a non-phosphorylating NADP+-dependentglyceraldehydes-3-phosphate dehydrogenase (GAPN) into yeast, either withor without the simultaneous knockout of GPD1. Bro, C., et al., Metab.Eng. 8:102-111 (2006); U.S. Patent Appl. Pub. No. US2006/0257983; Guo,Z. P., et al., Metab. Eng. 13:49-59 (2011). However, other cellularmechanisms exist to control the production and accumulation of glycerol,including glycerol exporters such as FPS1, that do not require theengineering of alternate NADP+/NADPH coupling or deletion of glycerolsynthesis genes. Tamas, M. J., et al., Mol. Microbiol. 31:1087-1004(1999).

FPS1 is a channel protein located in the plasma membrane that controlsthe accumulation and release of glycerol in yeast osmoregulation. Nullmutants of this strain accumulate large amounts of intracellularglycerol, grow much slower than wild-type, and consume the sugarsubstrate at a slower rate. Tamas, M. J., et al., Mol. Microbiol.31:1087-1004 (1999). Despite slower growth under anaerobic conditions,an fps1Δ strain can serve as an alternative to eliminatingNAD⁺-dependant glycerol activity. An fps1Δ strain has reduced glycerolformation yet has a completely functional NAD⁺-dependant glycerolsynthesis pathway. Alternatively, rather than deleting endogenous FPS1,constitutively active mutants of FPS 1 or homologs from other organismscan be used to regulate glycerol synthesis while keep the NAD⁺-dependantglycerol activity intact. In embodiments of the invention that modulateFPS1, the recombinant host cells can still synthesize and retainglycerol and achieve improved robustness relative to strains that areunable to make glycerol.

An example FPS1 sequence from S. cerevisiae is shown below.

S. cerevisiae FPS1 (nucleotide; coding sequence underlined; SEQ ID NO:1):

ttgacggcagttctcatagcatctcaaagcaatagcagtgcaaaagtacataaccgtaggaaggtacgcggtaggtatttgagttcgttggtggttatcctccgcaaggcgcttcggcggttatttgttgatagtcgaagaacaccaaaaaaaatgctgttattgctttctccgtaaacaataaaacccggtagcgggataacgcggctgatgcttttatttaggaaggaatacttacattatcatgagaacattgtcaagggcattctgatacgggccttccatcgcaagaaaaaggcagcaacggactgagggacggagagagttacggcataagaagtagtaggagagcagagtgtcataaagttatattattctcgtcctaaagtcaattagttctgttgcgcttgacaatatatgtcgtgtaataccgtcccttagcagaagaaagaaagacggatccatatatgttaaaatgcttcagagatgtttctttaatgtgccgtccaacaaaggtatcttctgtagcttcctctattttcgatcagatctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtgaatccggagacggcaagattgcccggccctttttgcggaaaagataaaacaagatatattgcactttttccaccaagaaaaacaggaagtggattaaaaaatcaacaaagtataacgcctattgtcccaataagcgtcggttgttcttctttattattttaccaagtacgctcgagggtacattctaatgcattaaaagacatgagtaatcctcaaaaagctctaaacgactttctgtccagtgaatctgttcatacacatgatagttctaggaaacaatctaataagcagtcatccgacgaaggacgctcttcatcacaaccttcacatcatcactctggtggtactaacaacaataataacaataataataataataataacagtaacaacaacaacaacggcaacgatgggggaaatgatgacgactatgattatgaaatgcaagattatagaccttctccgcaaagtgcgcggcctactcccacgtatgttccacaatattctgtagaaagtgggactgctttcccgattcaagaggttattcctagcgcatacattaacacacaagatataaaccataaagataacggtccgccgagtgcaagcagtaatagagcattcaggcctagagggcagaccacagtgtcggccaacgtgcttaacattgaagatttttacaaaaatgcagacgatgcgcataccatcccggagtcacatttatcgagaaggagaagtaggtcgagggctacgagtaatgctgggcacagtgccaatacaggcgccacgaatggcaggactactggtgcccaaactaatatggaaagcaatgaatcaccacgtaacgtccccattatggtgaagccaaagacattataccagaaccctcaaacacctacagtcttgccctccacataccatccaattaataaatggtcttccgtcaaaaacacttatttgaaggaatttttagccgagtttatgggaacaatggttatgattattttcggtagtgctgttgtttgtcaggtcaatgttgctgggaaaatacagcaggacaatttcaacgtggctttggataaccttaacgttaccgggtcttctgcagaaacgatagacgctatgaagagtttaacatccttggtttcatccgttgcgggcggtacctttgatgatgtggcattgggctgggctgctgccgtggtgatgggctatttctgcgctggtggtagtgccatctcaggtgctcatttgaatccgtctattacattagccaatttggtgtatagaggttttcccctgaagaaagttccttattactttgctggacaattgatcggtgccttcacaggcgctttgatcttgtttatttggtacaaaagggtgttacaagaggcatatagcgattggtggatgaatgaaagtgttgcgggaatgttttgcgtttttccaaagccttatctaagttcaggacggcaatttttttccgaatttttatgtggagctatgttacaagcaggaacatttgcgctgaccgatccttatacgtgtttgtcctctgatgttttcccattgatgatgtttattttgattttcattatcaatgcttccatggcttatcagacaggtacagcaatgaatttggctcgtgatctgggcccacgtcttgcactatatgcagttggatttgatcataaaatgctttgggtgcatcatcatcatttcttttgggttcccatggtaggcccatttattggtgcgttaatgggggggttggtttacgatgtctgtatttatcagggtcatgaatctccagtcaactggtctttaccagtttataaggaaatgattatgagagcctggtttagaaggcctggttggaagaagagaaatagagcaagaagaacatcggacctgagtgacttctcatacaataacgatgatgatgaggaatttggagaaagaatggctcttcaaaagacaaagaccaagtcatctatttcagacaacgaaaatgaagcaggagaaaagaaagtgcaatttaaatctgttcagcgcggcaaaagaacgtttggtggtataccaacaattcttgaagaagaagattccattgaaactgcttcgctaggtgcgacgacgactgattctattgggttatccgacacatcatcagaagattcgcattatggtaatgctaagaaggtaacatgagaaaacagacaagaaaaagaaacaaataatatagactgatagaaaaaaatactgcttactaccgccggtataatatatatatatatatatatttacatagatgattgcatagtgttttaaaaagctttcctaggttaagctatgaatcttcataacctaaccaactaaatatgaaaatactgacccatcgtcttaagtaagttgacatgaactcagcctggtcacctactatacatgatgtatcgcatggatggaaagaataccaaacgctaccttccaggttaatgatagtatccaaacctagttggaatttgccttgaacatcaagcagcgattcgatatcagttgggagcatcaatttggtcattggaataccatctatgcttttctcctcccatattcgcaaaagtagtaagggctcgttatatacttttgaatatgtaagatataattctatatgatttagtaatttattttctatacgctcagtatttttctgcagttgtcgagtaggtattaaacgcaaaagaagtccatccttttcatcattcaaatggacatcttggcaaagggcccagttatggaaaatctgggagtcatacaacgattgcagttggctatgccactcctggtaaggaatcatcaagtctgataattctgttttttagccctttttttttttttttcatggtgttctcttctcattgcttttcaattttaagttcgttacctttcatatagagtttcttaacagaaatttcacaacgaaaatataattaactacaggca

S. cerevisiae FPS1 (amino acid; SEQ ID NO:2):

Pyruvate Formate Lyase (PFL)

The conversion of the pyruvate to acetyl-CoA and formate is performed bypyruvate formate lyase (PFL). In E. coli, PFL is the primary enzymeresponsible for the production of formate. PFL is a dimer of PflB thatrequires the activating enzyme PflAE, which is encoded by pflA, radicalS-adenosylmethionine, and a single electron donor. See Waks, Z., andSilver, P. A., Appl. Env. Microbiol. 75:1867-1875 (2009). Waks andSilver engineered strains of S. cerevisiae to secrete formate by theaddition of PFL and AdhE from E. coli and deletion of endogenous formatedehydrogenases and to produce hydrogen in a two-step process using E.coli. Waks and Silver, however, did not combine formate production withthe removal of glycerol formation, and the use of formate as analternate electron acceptor for the reduction of glycerol was notproposed or evaluated.

PFL enzymes for use in the recombinant host cells of the invention cancome from a bacterial or eukaryotic source. Examples of bacterial PFLinclude, but are not limited to, Bacillus licheniformis DSM13, Bacilluslicheniformis ATCC14580, Streptococcus thermophilus CNRZ1066,Streptococcus thermophilus LMG18311, Streptococcus thermophilus LMD-9,Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_(—)2598),Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_(—)3313),Lactobacillus plantarum JDM1 (Gene Accession No. JDM1_(—)2695),Lactobacillus plantarum JDM1 (Gene Accession No. JDM1_(—)2087),Lactobacillus casei b123, Lactobacillus casei ATCC 334, Bifidobacteriumadolescentis, Bifidobacterium longum NCC2705, Bifidobacterium longumDJO10A, Bifidobacterium animalis DSM 10140, Clostridium cellulolyticum,or Escherichia coli. Additional PFL enzymes may be from the PFL1 family,the RNR pfl superfamily, or the PFL2 superfamily.

pflA sequences from bacteria include:

-   -   Bacillus licheniformis DSM13 (nucleotide; SEQ ID NO:3):    -   Bacillus licheniformis DSM13 (amino acid; SEQ ID NO:4):    -   Bacillus licheniformis ATCC14580 (nucleotide; SEQ ID NO:5):    -   Bacillus licheniformis ATCC14580 (amino acid; SEQ ID NO:6):    -   Streptococcus thermophilus CNRZ1066 (nucleotide; SEQ ID NO:7):    -   Streptococcus thermophilus CNRZ1066 (amino acid; SEQ ID NO:8):    -   Streptococcus thermophilus LMG18311 (nucleotide; SEQ ID NO:9):    -   Streptococcus thermophilus LMG18311 (amino acid; SEQ ID NO:10):    -   Streptococcus thermophilus LMD-9 (nucleotide; SEQ ID NO:11):    -   Streptococcus thermophilus LMD-9 (amino acid; SEQ ID NO:12):    -   Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_(—)2596)        (nucleotide; SEQ ID NO:13):    -   Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_(—)2596)        (amino acid; SEQ ID NO:14):    -   Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_(—)3314)        (nucleotide; SEQ ID NO:15):    -   Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_(—)3314)        (amino acid; SEQ ID NO:16):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2660)        (nucleotide; SEQ ID NO:17):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2660)        (amino acid; SEQ ID NO:18):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2085)        (nucleotide; SEQ ID NO:19):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2085)        (amino acid; SEQ ID NO:20):    -   Lactobacillus casei b123 (nucleotide; SEQ ID NO:21):    -   Lactobacillus casei b123 (amino acid; SEQ ID NO:22):    -   Lactobacillus casei ATCC 334 (nucleotide; SEQ ID NO:23):    -   Lactobacillus casei ATCC 334 (amino acid; SEQ ID NO:24):    -   Bifidobacterium adolescentis (nucleotide; SEQ ID NO:25):    -   Bifidobacterium adolescentis (amino acid; SEQ ID NO:26):    -   Bifidobacterium longum NCC2705 (nucleotide; SEQ ID NO:27):    -   Bifidobacterium longum NCC2705 (amino acid; SEQ ID NO:28):    -   Bifidobacterium longum DJO10A (nucleotide; SEQ ID NO:29):    -   Bifidobacterium longum DJO10A (amino acid; SEQ ID NO:30):    -   Bifidobacterium animalis DSM 10140 (nucleotide; SEQ ID NO:31):    -   Bifidobacterium animalis DSM 10140 (amino acid; SEQ ID NO:32):    -   Clostridium cellulolyticum (nucleotide; SEQ ID NO:33):    -   Clostridium cellulolyticum (amino acid; SEQ ID NO:34):    -   Escherichia coli (nucleotide; SEQ ID NO:35):    -   Escherichia coli (amino acid; SEQ ID NO:36):

pflB sequences from bacteria include:

-   -   Bacillus licheniformis DSM13 (nucleotide; SEQ ID NO:37):    -   Bacillus licheniformis DSM13 (amino acid; SEQ ID NO:38):    -   Bacillus licheniformis ATCC14580 (nucleotide; SEQ ID NO:39):    -   Bacillus licheniformis ATCC14580 (amino acid; SEQ ID NO:40):    -   Streptococcus thermophilus CNRZ1066 (nucleotide; SEQ ID NO:41):    -   Streptococcus thermophilus CNRZ1066 (amino acid; SEQ ID NO:42):    -   Streptococcus thermophilus LMG18311 (nucleotide; SEQ ID NO:43):    -   Streptococcus thermophilus LMG18311 (amino acid; SEQ ID NO:44):    -   Streptococcus thermophilus LMD-9 (nucleotide; SEQ ID NO:45):    -   Streptococcus thermophilus LMD-9 (amino acid; SEQ ID NO:46):    -   Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_(—)2598)        (nucleotide; SEQ ID NO:47):    -   Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_(—)2598)        (amino acid; SEQ ID NO:48):    -   Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_(—)3313)        (nucleotide; SEQ ID NO:49):    -   Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_(—)3313)        (amino acid; SEQ ID NO:50):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2695)        (nucleotide; SEQ ID NO:51):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2695)        (amino acid; SEQ ID NO:52):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2087)        (nucleotide; SEQ ID NO:53):    -   Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_(—)2087)        (amino acid; SEQ ID NO:54):    -   Lactobacillus casei b123 (nucleotide; SEQ ID NO:55):    -   Lactobacillus casei b123 (amino acid; SEQ ID NO:56):    -   Lactobacillus casei ATCC 334 (nucleotide; SEQ ID NO:57):    -   Lactobacillus casei ATCC 334 (amino acid; SEQ ID NO:58):    -   Bifidobacterium adolescentis (nucleotide; SEQ ID NO:59):    -   Bifidobacterium adolescentis (amino acid; SEQ ID NO:60):    -   Bifidobacterium longum NCC2705 (nucleotide; SEQ ID NO:61):    -   Bifidobacterium longum NCC2705 (amino acid; SEQ ID NO:62):    -   Bifidobacterium longum DJO10A (nucleotide; SEQ ID NO:63):    -   Bifidobacterium longum DJO10A (amino acid; SEQ ID NO:64):    -   Bifidobacterium animalis DSM 10140 (nucleotide; SEQ ID NO:65):    -   Bifidobacterium animalis DSM 10140 (amino acid; SEQ ID NO:66):    -   Clostridium cellulolyticum (nucleotide; SEQ ID NO:67):    -   Clostridium cellulolyticum (amino acid; SEQ ID NO:68):    -   Escherichia coli (nucleotide; SEQ ID NO:69):    -   Escherichia coli (amino acid; SEQ ID NO:70):

Examples of eukaryotic PFL include, but are not limited to,Chlamydomonas reinhardtii PflA1, Piromyces sp. E2, or Neocallimastixfrontalis, Acetabularia acetabulum, Haematococcus pluvialis, Volvoxcarteri, Ostreococcus tauri, Ostreococcus lucimarinus, Micromonaspusilla, Micromonas sp., Porphyra haitanensis, and Cyanophora paradoxa),an opisthokont (Amoebidium parasiticum), an amoebozoan (Mastigamoebabalamuthi), a stramenopile (Thalassiosira pseudonana (2)) and ahaptophyte (Prymnesium parvum), M. pusilla, Micromonas sp. O. tauri andO. lucimarinus) an amoebozoan (M. balamuthi), and a stramenopile (T.pseudonana). See Stairs, C. W., et al., “Eukaryotic pyruvate formatelyase and its activating enzyme were acquired laterally from afirmicute,” Mol. Biol. and Evol., published on-line on Feb. 3, 2011, athttp://mbe.oxfordjournals.org/.

pflA sequences from eukaryotes include:

-   -   Chlamydomonas reinhardtii PflA1 (nucleotide; SEQ ID NO:71):    -   Chlamydomonas reinhardtii PflA1 (amino acid; SEQ ID NO:72):    -   Neocallimastix frontalis (nucleotide; SEQ ID NO:73):    -   Neocallimastix frontalis (amino acid; SEQ ID NO:74):

pfl1 sequences from eukaryotes include:

-   -   Chlamydomonas reinhardtii PflA (nucleotide; SEQ ID NO:75):    -   Chlamydomonas reinhardtii PflA (amino acid; SEQ ID NO:76):    -   Piromyces sp. E2 (nucleotide; SEQ ID NO:77):    -   Piromyces sp. E2 (amino acid; SEQ ID NO:78):    -   Neocallimastix frontalis (nucleotide—partial CDS, missing start;        SEQ ID NO:79):    -   Neocallimastix frontalis (amino acid—partial CDS, missing start;        SEQ ID NO:80):        Acetaldehyde/Alcohol Dehydrogenases

Engineering of acetaldehyde dehydrogenases, alcohol dehydrogenases,and/or bifunctional acetylaldehyde/alcohol dehydrogenases into a cellcan increase the production of ethanol. However, because the productionof ethanol is redox neutral, an acetaldehyde/alcohol dehydrogenaseactivity cannot serve as an alternative for the redox balancing that theproduction of glycerol provides to a cell in anaerobic metabolism. WhenMedina attempted to express an acetylaldehyde dehydrogenase, mhpF, fromE. coli in an S. cerevisiae gpd1/2 deletion strain, the strain did notgrow under anaerobic conditions in the presence of glucose as the solesource of carbon. Medina, V. G., et al., Appl. Env. Microbiol.76:190-195 (2010); see also EP 2277989. Rather, the anaerobic growth ofthe glycerol-deletion strain required the presence of acetic acid.However, an acetylaldehyde dehydrogenase has not been expressed incombination with PFL or with the recombinant host cells of theinvention. Additionally, replacing the endogenous acetylaldehydedehydrogenase activity with either an improved acetaldehydedehydrogenase or using a bifunctional acetaldehyde/alcohol dehydrogenase(AADH) can positively affect the in vivo kinetics of the reactionproviding for improved growth of the host strain.

AADH enzymes for use in the recombinant host cells of the invention cancome from a bacterial or eukaryotic source. Examples of bacterial AADHinclude, but are not limited to, Clostridium phytofermentans,Escherichia coli, Bacillus coagulans, Bacillus lentus, Bacilluslicheniformis, Bacillus pumilus, Bacillus subtilis, Bacteroidesamylophilus, Bacteroides capillosus, Bacteroides ruminocola, Bacteroidessuis, Bifidobacterium adolescentis, Bifidobacterium animalis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlongum, Bifidobacterium thermophilum, Lactobacillus acidophilus,Lactobacillus brevis, Lactobacillus buchneri (cattle only),Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii,Lactobacillus farciminis (swine only), Lactobacillus fermentum,Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum,Lactobacillus reuterii, Leuconostoc mesenteroides, Pediococcusacidilacticii, Pediococcus pentosaceus, Propionibacterium acidpropionici(cattle only), Propionibacterium freudenreichii, Propionibacteriumshermanii, Enterococcus cremoris, Enterococcus diacetylactis,Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis, orEnterococcus thermophilus

AdhE bacterial sequences include:

-   -   Clostridium phytofermentans (nucleotide; SEQ ID NO:81):    -   Clostridium phytofermentans (amino acid; SEQ ID NO:82):    -   Escherichia coli (nucleotide; SEQ ID NO:83):    -   Escherichia coli (amino acid; SEQ ID NO:84):    -   Bifidobacterium adolescentis (amino acid; SEQ ID NO:100):    -   Bacillus coagulans (amino acid; SEQ ID NO:101):    -   Bacillus licheniformis (amino acid; SEQ ID NO: 102):    -   Enterococcus faecium TX1330 (amino acid; SEQ ID NO:103):

Examples of eukaryotic AdhE include, but are not limited to,Chlamydomonas reinhardtii AdhE, Piromyces sp. E2, or Neocallimastixfrontalis.

AdhE sequences from eukaryotes include:

-   -   Chlamydomonas reinhardtii AdhE (nucleotide; SEQ ID NO:85):    -   Chlamydomonas reinhardtii AdhE (amino acid; SEQ ID NO:86):    -   Piromyces sp. E2 (nucleotide; SEQ ID NO:87):    -   Piromyces sp. E2 (amino acid; SEQ ID NO:88):        Consolidated Bioprocessing

Consolidated bioprocessing (CBP) is a processing strategy for cellulosicbiomass that involves consolidating into a single process step fourbiologically-mediated events: enzyme production, hydrolysis, hexosefermentation, and pentose fermentation. Implementing this strategyrequires development of microorganisms that both utilize cellulose,hemicellulosics, and other biomass components while also producing aproduct of interest at sufficiently high yield and concentrations. Thefeasibility of CBP is supported by kinetic and bioenergetic analysis.See van Walsum and Lynd (1998) Biotech. Bioeng. 58:316.

CBP offers the potential for lower cost and higher efficiency thanprocesses featuring dedicated saccharolytic enzyme production. Thebenefits result in part from avoided capital costs, substrate and otherraw materials, and utilities associated with saccharolytic enzymeproduction. In addition, several factors support the realization ofhigher rates of hydrolysis, and hence reduced reactor volume and capitalinvestment using CBP, including enzyme-microbe synergy and the use ofthermophilic organisms and/or complexed saccharolytic systems. Moreover,cellulose-adherent cellulolytic microorganisms are likely to competesuccessfully for products of cellulose hydrolysis with non-adheredmicrobes, e.g., contaminants, which could increase the stability ofindustrial processes based on microbial cellulose utilization. Progressin developing CBP-enabling microorganisms is being made through twostrategies: engineering naturally occurring saccharolytic microorganismsto improve product-related properties, such as yield and titer; andengineering non-saccharolytic organisms that exhibit high product yieldsand titers to express a heterologous saccharolytic enzyme systemenabling starch, cellulose, and, hemicellulose utilization.

Starch and Cellulose Degradation

The degradation of starch into component sugar units proceeds viaamylolytic enzymes. Amylase is an example of an amylolytic enzyme thatis present in human saliva, where it begins the chemical process ofdigestion. The pancreas also makes amylase (alpha amylase) to hydrolyzedietary starch into disaccharides and trisaccharides which are convertedby other enzymes to glucose to supply the body with energy. Plants andsome bacteria also produce amylases. Amylases are glycoside hydrolasesand act on α-1,4-glycosidic bonds.

Several amylolytic enzymes are implicated in starch hydrolysis.Alpha-amylases (EC 3.2.1.1) (alternate names: 1,4-α-D-glucanglucanohydrolase; glycogenase) are calcium metalloenzymes, i.e.,completely unable to function in the absence of calcium. By acting atrandom locations along the starch chain, alpha-amylase breaks downlong-chain carbohydrates, ultimately yielding maltotriose and maltosefrom amylose, or maltose, glucose and “limit dextrin” from amylopectin.Because it can act anywhere on the substrate, alpha-amylase tends to befaster-acting than beta-amylase. Another form of amylase, beta-amylase(EC 3.2.1.2) (alternate names: 1,4-α-D-glucan maltohydrolase;glycogenase; saccharogen amylase) catalyzes the hydrolysis of the secondα-1,4 glycosidic bond, cleaving off two glucose units (maltose) at atime. The third amylase is gamma-amylase (EC 3.2.1.3) (alternate names:Glucan 1,4-α-glucosidase; amyloglucosidase; Exo-1,4-α-glucosidase;glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase).In addition to cleaving the last α(1-4)glycosidic linkages at thenonreducing end of amylose and amylopectin, yielding glucose,gamma-amylase will cleave α(1-6) glycosidic linkages.

A fourth enzyme, alpha-glucosidase, acts on maltose and other shortmalto-oligosaccharides produced by alpha-, beta-, and gamma-amylases,converting them to glucose.

Three major types of enzymatic activities degrade native cellulose. Thefirst type is endoglucanases (1,4-f3-D-glucan 4-glucanohydrolases; EC3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharidechain of amorphous cellulose, generating oligosaccharides of varyinglengths and consequently new chain ends. The second type areexoglucanases, including cellodextrinases (1,4-β-D-glucanglucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-β-D-glucancellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processivemanner on the reducing or non-reducing ends of cellulose polysaccharidechains, liberating either glucose (glucanohydrolases) or cellobiose(cellobiohydrolase) as major products. Exoglucanases can also act onmicrocrystalline cellulose, presumably peeling cellulose chains from themicrocrystalline structure. The third type are β-glucosidases(β-glucoside glucohydrolases; EC 3.2.1.21). β-Glucosidases hydrolyzesoluble cellodextrins and cellobiose to glucose units.

Even though yeast strains expressing enzymes for the production of fuelethanol from whole grain or starch have been previously disclosed, theapplication has not been commercialized in the grain-based fuel ethanolindustry, due to the relatively poor ability of the resulting strains toproduce/tolerate high levels of ethanol. For example, U.S. Pat. No.7,226,776 discloses that a polysaccharase enzyme expressing ethanologencan make ethanol directly from carbohydrate polymers, but the maximalethanol titer demonstrated is 3.9 g/l. U.S. Pat. No. 5,422,267 disclosesthe use of a glucoamylase in yeast for production of alcoholicbeverages; however, no commercially relevant titers of ethanol aredisclosed.

Heterologous Saccharolytic Enzymes

According to one aspect of the present invention, the expression ofheterologous saccharolytic enzymes the recombinant microorganisms of theinvention can be used advantageously to produce products such as ethanolfrom biomass sources. For example, cellulases from a variety of sourcescan be heterologously expressed to successfully increase efficiency ofethanol production. The saccharolytic enzymes can be from fungi, yeast,bacteria, plant, protozoan or termite sources. In some embodiments, thesaccharolytic enzyme is from H. grisea, T. aurantiacus, T. emersonii, T.reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis,M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus,Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum,Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagusdegradans, Piromyces equii, Neocallimastix patricarum or Arabidopsisthaliana.

In some embodiments, the cellulase for expression in the recombinantmicroorganisms of the invention is any cellulase disclosed in Table 4 orTable 7 in copending International Appl. No. PCT/US2011/039192,incorporated by reference herein, or any cellulase suitable forexpression in an appropriate host cell. In other embodiments, theamylase for expression in the recombinant microorganisms of theinvention is any amylase such as alpha-amylases, beta-amylases,glucoamylases, alpha-glucosidases, pullulanase, or isopullulanaseparalogues or orthologues, any amylase disclosed in Tables 15-19,preferably in Table 19, in copending International Appl. No.PCT/US2011/039192, incorporated by reference herein, or any amylasesuitable for expression in an appropriate host cell. In some embodimentsof the invention, multiple saccharolytic enzymes from a single organismare co-expressed in the same recombinant microorganism. In someembodiments of the invention, multiple saccharolytic enzymes fromdifferent organisms are co-expressed in the same recombinantmicroorganism. In particular, saccharolytic enzymes from two, three,four, five, six, seven, eight, nine or more organisms can beco-expressed in the same recombinant microorganism. Similarly, theinvention can encompass co-cultures of yeast strains, wherein the yeaststrains express different saccharolytic enzymes. Co-cultures can includeyeast strains expressing heterologous saccharolytic enzymes from thesame organisms or from different organisms. Co-cultures can includeyeast strains expressing saccharolytic enzymes from two, three, four,five, six, seven, eight, nine or more organisms.

Lignocellulases for expression in the recombinant microorganisms of thepresent invention include both endoglucanases and exoglucanases. Otherlignocellulases for expression in the recombinant microorganisms of theinvention include accesory enzymes which can act on the lignocellulosicmaterial. The lignocellulases can be, for example, endoglucanases,glucosidases, cellobiohydrolases, xylanases, glucanases, xylosidases,xylan esterases, arabinofuranosidases, galactosidases, cellobiosephosphorylases, cellodextrin phosphorylases, mannanases, mannosidases,xyloglucanases, endoxylanases, glucuronidases, acetylxylanesterases,arabinofuranohydrolases, swollenins, glucuronyl esterases, expansins,pectinases, and feruoyl esterases. In some embodiments, thelignocellulases of the invention can be any suitable enzyme fordigesting the desired lignocellulosic material.

In certain embodiments of the invention, the lignocellulase can be anendoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase,xylosidase, xylan esterase, arabinofuranosidase, galactosidase,cellobiose phosphorylase, cellodextrin phosphorylase, mannanase,mannosidase, xyloglucanase, endoxylanase, glucuronidase,acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronylesterase, expansin, pectinase, and feruoyl esterase paralogue ororthologue. In particular embodiments, the lignocellulase is derivedfrom any species named in Tables 4 and 7, in copending InternationalAppl. No. PCT/US2011/039192, incorporated by reference herein.

Xylose Metabolism

Xylose is a five-carbon monosaccharide that can be metabolized intouseful products by a variety of organisms. There are two main pathwaysof xylose metabolism, each unique in the characteristic enzymes theyutilize. One pathway is called the “Xylose Reductase-XylitolDehydrogenase” or XR-XDH pathway. Xylose reductase (XR) and xylitoldehydrogenase (XDH) are the two main enzymes used in this method ofxylose degradation. XR, encoded by the XYL1 gene, is responsible for thereduction of xylose to xylitol and is aided by cofactors NADH or NADPH.Xylitol is then oxidized to xylulose by XDH, which is expressed throughthe XYL2 gene, and accomplished exclusively with the cofactor NAD⁺.Because of the varying cofactors needed in this pathway and the degreeto which they are available for usage, an imbalance can result in anoverproduction of xylitol byproduct and an inefficient production ofdesirable ethanol. Varying expression of the XR and XDH enzyme levelshave been tested in the laboratory in the attempt to optimize theefficiency of the xylose metabolism pathway.

The other pathway for xylose metabolism is called the “Xylose Isomerase”(XI) pathway. Enzyme XI is responsible for direct conversion of xyloseinto xylulose, and does not proceed via a xylitol intermediate. Bothpathways create xylulose, although the enzymes utilized are different.After production of xylulose both the XR-XDH and XI pathways proceedthrough the enzyme xylulokinase (XK), encoded on gene XKS1, to furthermodify xylulose into xylulose-5-phosphate where it then enters thepentose phosphate pathway for further catabolism.

Studies on flux through the pentose phosphate pathway during xylosemetabolism have revealed that limiting the speed of this step may bebeneficial to the efficiency of fermentation to ethanol. Modificationsto this flux that may improve ethanol production include a) loweringphosphoglucose isomerase activity, b) deleting the GND1 gene, and c)deleting the ZWF1 gene (Jeppsson et al., Appl. Environ. Microbiol.68:1604-09 (2002)). Since the pentose phosphate pathway producesadditional NADPH during metabolism, limiting this step will help tocorrect the already evident imbalance between NAD(P)H and NAD⁺ cofactorsand reduce xylitol byproduct. Another experiment comparing the twoxylose metabolizing pathways revealed that the XI pathway was best ableto metabolize xylose to produce the greatest ethanol yield, while theXR-XDH pathway reached a much faster rate of ethanol production(Karhumaa et al., Microb Cell Fact. 2007 Feb. 5; 6:5). See alsoInternational Publication No. WO2006/009434, incorporated herein byreference in its entirety.

In some embodiments, the recombinant microorganisms of the inventionhave the ability to metabolize xylose using one or more of the aboveenzymes.

Arabinose Metabolism

Arabinose is a five-carbon monosaccharide that can be metabolized intouseful products by a variety of organisms. L-Arabinose residues arefound widely distributed among many heteropolysaccharides of differentplant tissues, such as arabinans, arabinogalactans, xylans andarabinoxylans. Bacillus species in the soil participate in the earlystages of plant material decomposition, and B. subtilis secretes threeenzymes, an endo-arabanase and two arabinosidases, capable of releasingarabinosyl oligomers and L-arabinose from plant cell.

Three pathways for L-arabinose metabolism in microorganisms have beendescribed. Many bacteria, including Escherichia coli, use arabinoseisomerase (AraA; E.C. 5.3.1.4), ribulokinase (AraB; E.C. 2.7.1.16), andribulose phosphate epimerase (AraD; E.C. 5.1.3.4) to sequentiallyconvert L-arabinose to D-xylulose-5-phosphate through L-ribulose andL-ribulose 5-phosphate. See, e.g., Sa-Nogueira I, et al., Microbiology143:957-69 (1997). The D-xylulose-5-phosphate then enters the pentosephosphate pathway for further catabolism. In the second pathway,L-arabinose is converted to L-2-keto-3-deoxyarabonate (L-KDA) by theconsecutive action of enzymes arabinose dehydrogenase (ADH),arabinolactone (AL), and arabinonate dehydratase (AraC). See, e.g.,Watanabe, S, et al., J. Biol. Chem. 281: 2612-2623 (2006). L-KDA can befurther metabolized in two alternative pathways: 1) L-KDA conversion to2-ketoglutarate via 2-ketoglutaric semialdehyde (KGSA) by L-KDAdehydratase and KGSA dehydrogenase or 2) L-KDA conversion to pyruvateand glycolaldehyde by L-KDA aldolase. In the third, fungal pathway,L-arabinose is converted to D-xylulose-5-phosphate through L-arabinitol,L-xylulose, and xylitol, by enzymes such as NAD(P)H-dependent aldosereductase (AR), L-arabinitol 4-dehydrogenase (ALDH), L-xylulosereductase (LXR), xylitol dehydrogenase (XylD), and xylulokinase (XylB).These, and additional proteins involved in arabinose metabolism andregulation may be found athttp://www.nmpdr.org/FIG/wiki/rest.cgi/NmpdrPlugin/SeedViewer?page=Subsystems;subsystem=L-Arabinose_utilization, visited Mar. 21, 2011, which isincorporated by reference herein in its entirety.

AraC protein regulates expression of its own synthesis and the othergenes of the Ara system. See Schleif, R., Trends Genet. 16(12):559-65(2000). In the E. coli, the AraC protein positively and negativelyregulates expression of the proteins required for the uptake andcatabolism of the sugar L-arabinose. Homologs of AraC, such asregulatory proteins RhaR and RhaS of the rhamnose operon, have beenidentified that contain regions homologous to the DNA-binding domain ofAraC (Leal, T. F. and de Sa-Nogueira, I., FEMS Microbiol Lett.241(1):41-48 (2004)). Such arabinose regulatory proteins are referred toas the AraC/XylS family. See also, Mota, L. J., et al., Mol. Microbiol.33(3):476-89 (1999); Mota, L. J., et al., J Bacteriol. 183(14):4190-201(2001).

In E. coli, the transport of L-arabinose across the E. coli cytoplasmicmembrane requires the expression of either the high-affinity transportoperon, araFGH, a binding protein-dependent system on the low-affinitytransport operon, araE, a proton symporter. Additional arabinosetransporters include those identified from K. marxianus and P.guilliermondii, disclosed in U.S. Pat. No. 7,846,712, which isincorporated by reference herein.

In some embodiments, the recombinant microorganisms of the inventionhave the ability to metabolize arabinose using one or more of the aboveenzymes.

Microorganisms

The present invention includes multiple strategies for the developmentof microorganisms with the combination of substrate-utilization andproduct-formation properties required for CBP. The “native cellulolyticstrategy” involves engineering naturally occurring cellulolyticmicroorganisms to improve product-related properties, such as yield andtiter. The “recombinant cellulolytic strategy” involves engineeringnatively non-cellulolytic organisms that exhibit high product yields andtiters to express a heterologous cellulase system that enables celluloseutilization or hemicellulose utilization or both.

Many bacteria have the ability to ferment simple hexose sugars into amixture of acidic and pH-neutral products via the process of glycolysis.The glycolytic pathway is abundant and comprises a series of enzymaticsteps whereby a six carbon glucose molecule is broken down, via multipleintermediates, into two molecules of the three carbon compound pyruvate.This process results in the net generation of ATP (biological energysupply) and the reduced cofactor NADH.

Pyruvate is an important intermediary compound of metabolism. Forexample, under aerobic conditions pyruvate may be oxidized to acetylcoenzyme A (acetyl-CoA), which then enters the tricarboxylic acid cycle(TCA), which in turn generates synthetic precursors, CO₂, and reducedcofactors. The cofactors are then oxidized by donating hydrogenequivalents, via a series of enzymatic steps, to oxygen resulting in theformation of water and ATP. This process of energy formation is known asoxidative phosphorylation.

Under anaerobic conditions (no available oxygen), fermentation occurs inwhich the degradation products of organic compounds serve as hydrogendonors and acceptors. Excess NADH from glycolysis is oxidized inreactions involving the reduction of organic substrates to products,such as lactate and ethanol. In addition, ATP is regenerated from theproduction of organic acids, such as acetate, in a process known assubstrate level phosphorylation. Therefore, the fermentation products ofglycolysis and pyruvate metabolism include a variety of organic acids,alcohols and CO₂.

Most facultative anaerobes metabolize pyruvate aerobically via pyruvatedehydrogenase (PDH) and the tricarboxylic acid cycle (TCA). Underanaerobic conditions, the main energy pathway for the metabolism ofpyruvate is via pyruvate-formate-lyase (PFL) pathway to give formate andacetyl-CoA. Acetyl-CoA is then converted to acetate, viaphosphotransacetylase (PTA) and acetate kinase (ACK) with theco-production of ATP, or reduced to ethanol via acetalaldehydedehydrogenase (ACDH) and alcohol dehydrogenase (ADH). In order tomaintain a balance of reducing equivalents, excess NADH produced fromglycolysis is re-oxidized to NAD⁺ by lactate dehydrogenase (LDH) duringthe reduction of pyruvate to lactate. NADH can also be re-oxidized byACDH and ADH during the reduction of acetyl-CoA to ethanol, but this isa minor reaction in cells with a functional LDH.

Host Cells

Host cells useful in the present invention include any prokaryotic oreukaryotic cells; for example, microorganisms selected from bacterial,algal, and yeast cells. Among host cells thus suitable for the presentinvention are microorganisms, for example, of the genera Aeromonas,Aspergillus, Bacillus, Escherichia, Kluyveromyces, Pichia, Rhodococcus,Saccharomyces and Streptomyces.

In some embodiments, the host cells are microorganisms. In oneembodiment the microorganism is a yeast. According to the presentinvention the yeast host cell can be, for example, from the generaSaccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces,Hansenula, Kloeckera, Schwanniomyces, and Yarrowia. Yeast species ashost cells may include, for example, S. cerevisiae, S. bulderi, S.barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K.marxianus, or K. fragilis. In some embodiments, the yeast is selectedfrom the group consisting of Saccharomyces cerevisiae,Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichiastipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma,Candida utilis, Arxula adeninivorans, Debaryomyces hansenii,Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomycesoccidentalis. In one particular embodiment, the yeast is Saccharomycescerevisiae. In another embodiment, the yeast is a thermotolerantSaccharomyces cerevisiae. The selection of an appropriate host is deemedto be within the scope of those skilled in the art from the teachingsherein.

In some embodiments, the host cell is an oleaginous cell. The oleaginoushost cell can be an oleaginous yeast cell. For example, the oleaginousyeast host cell can be from the genera Blakeslea, Candida, Cryptococcus,Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium,Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. According to thepresent invention, the oleaginous host cell can be an oleaginousmicroalgae host cell. For example, the oleaginous microalgea host cellcan be from the genera Thraustochytrium or Schizochytrium. Biodieselcould then be produced from the triglyceride produced by the oleaginousorganisms using conventional lipid transesterification processes. Insome particular embodiments, the oleaginous host cells can be induced tosecrete synthesized lipids. Embodiments using oleaginous host cells areadvantegeous because they can produce biodiesel from lignocellulosicfeedstocks which, relative to oilseed substrates, are cheaper, can begrown more densely, show lower life cycle carbon dioxide emissions, andcan be cultivated on marginal lands.

In some embodiments, the host cell is a thermotolerant host cell.Thermotolerant host cells can be particularly useful in simultaneoussaccharification and fermentation processes by allowing externallyproduced cellulases and ethanol-producing host cells to performoptimally in similar temperature ranges.

Thermotolerant host cells can include, for example, Issatchenkiaorientalis, Pichia mississippiensis, Pichia mexicana, Pichia farinosa,Clavispora opuntiae, Clavispora lusitaniae, Candida mexicana, Hansenulapolymorpha and Kluyveromyces host cells. In some embodiments, thethermotolerant cell is an S. cerevisiae strain, or other yeast strain,that has been adapted to grow in high temperatures, for example, byselection for growth at high temperatures in a cytostat.

In some particular embodiments, the host cell is a Kluyveromyces hostcell. For example, the Kluyveromyces host cell can be a K. lactis, K.marxianus, K. blattae, K. phaffii, K. yarrowii, K. aestuarii, K.dobzhanskii, K. wickerhamii K. thermotolerans, or K. waltii host cell.In one embodiment, the host cell is a K. lactis, or K. marxianus hostcell. In another embodiment, the host cell is a K. marxianus host cell.

In some embodiments, the thermotolerant host cell can grow attemperatures above about 30° C., about 31° C., about 32° C., about 33°C., about 34° C., about 35° C., about 36° C., about 37° C., about 38°C., about 39° C., about 40° C., about 41° C. or about 42° C. In someembodiments of the present invention the thermotolerant host cell canproduce ethanol from cellulose at temperatures above about 30° C., about31° C., about 32° C., about 33° C., about 34° C., about 35° C., about36° C., about 37° C., about 38° C., about 39° C., about 40° C., about41° C., about 42° C., or about 43° C., or about 44° C., or about 45° C.,or about 50° C.

In some embodiments of the present invention, the thermotolerant hostcell can grow at temperatures from about 30° C. to 60° C., about 30° C.to 55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C.to 55° C. or about 40° C. to 50° C. In some embodiments of the presentinvention, the thermotolterant host cell can produce ethanol fromcellulose at temperatures from about 30° C. to 60° C., about 30° C. to55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C. to55° C. or about 40° C. to 50° C.

In some embodiments, the host cell has the ability to metabolize xylose.Detailed information regarding the development of the xylose-utilizingtechnology can be found in the following publications: Kuyper M et al.FEMS Yeast Res. 4: 655-64 (2004), Kuyper M et al. FEMS Yeast Res.5:399-409 (2005), and Kuyper M et al. FEMS Yeast Res. 5:925-34 (2005),which are herein incorporated by reference in their entirety. Forexample, xylose-utilization can be accomplished in S. cerevisiae byheterologously expressing the xylose isomerase gene, XylA, e.g., fromthe anaerobic fungus Piromyces sp. E2, overexpressing five S. cerevisiaeenzymes involved in the conversion of xylulose to glycolyticintermediates (xylulokinase, ribulose 5-phosphate isomerase, ribulose5-phosphate epimerase, transketolase and transaldolase) and deleting theGRE3 gene encoding aldose reductase to minimise xylitol production.

In some embodiments, the host cell has the ability to metabolizearabinose. For example, arabinose-utilization can be accomplished byheterologously expressing, e.g., one or more of arabinose isomerase,ribulokinase, or ribulose phosphate epimerase.

The host cells can contain antibiotic markers or can contain noantibiotic markers.

In certain embodiments, the host cell is a microorganism that is aspecies of the genera Thermoanaerobacterium, Thermoanaerobacter,Clostridium, Geobacillus, Saccharococcus, Paenibacillus, Bacillus,Caldicellulosiruptor, Anaerocellum, or Anoxybacillus. In certainembodiments, the host cell is a bacterium selected from the groupconsisting of: Thermoanaerobacterium thermosulfurigenes,Thermoanaerobacterium aotearoense, Thermoanaerobacteriumpolysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacteriumxylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobiumbrockii, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterthermohydrosulfuricus, Thermoanaerobacter ethanolicus,Thermoanaerobacter brocki, Clostridium thermocellum, Clostridiumcellulolyticum, Clostridium phytofermentans, Clostridiumstraminosolvens, Geobacillus thermoglucosidasius, Geobacillusstearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccusthermophilus, Paenibacillus campinasensis, Bacillus flavothermus,Anoxybacillus kamchatkensis, Anoxybacillus gonensis,Caldicellulosiruptor acetigenus, Caldicellulosiruptor saccharolyticus,Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis,Caldicellulosiruptor lactoaceticus, and Anaerocellum thermophilum. Incertain embodiments, the host cell is Clostridium thermocellum,Clostridium cellulolyticum, or Thermoanaerobacterium saccharolyticum.

Codon Optimized Polynucleotides

The polynucleotides encoding heterologous cellulases can becodon-optimized. As used herein the term “codon-optimized coding region”means a nucleic acid coding region that has been adapted for expressionin the cells of a given organism by replacing at least one, or more thanone, or a significant number, of codons with one or more codons that aremore frequently used in the genes of that organism.

In general, highly expressed genes in an organism are biased towardscodons that are recognized by the most abundant tRNA species in thatorganism. One measure of this bias is the “codon adaptation index” or“CAI,” which measures the extent to which the codons used to encode eachamino acid in a particular gene are those which occur most frequently ina reference set of highly expressed genes from an organism.

The CAI of codon optimized sequences of the present inventioncorresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, orabout 1.0. A codon optimized sequence may be further modified forexpression in a particular organism, depending on that organism'sbiological constraints. For example, large runs of “As” or “Ts” (e.g.,runs greater than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can beremoved from the sequences if these are known to effect transcriptionnegatively. Furthermore, specific restriction enzyme sites may beremoved for molecular cloning purposes. Examples of such restrictionenzyme sites include Pad, AscI, BamHI, BglII, EcoRI and XhoI.Additionally, the DNA sequence can be checked for direct repeats,inverted repeats and mirror repeats with lengths of ten bases or longer,which can be modified manually by replacing codons with “second best”codons, i.e., codons that occur at the second highest frequency withinthe particular organism for which the sequence is being optimized.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG Ter TGG Trp (W)C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro(P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg(R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACTThr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGCSer (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met ACG Thr(T) AAG Lys (K) AGG Arg (R) (M) G GTT Val (V) GCT Ala (A) GAT Asp (D)GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V)GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E)GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at http://www.kazusa.or.jp/codon/ (visited Feb.28, 2011), and these tables can be adapted in a number of ways. SeeNakamura, Y., et al. “Codon usage tabulated from the international DNAsequence databases: status for the year 2000,” Nucl. Acids Res. 28:292(2000). Codon usage tables for yeast, calculated from GenBank Release128.0 [15 Feb. 2002], are reproduced below as Table 2. This table usesmRNA nomenclature, and so instead of thymine (T) which is found in DNA,the tables use uracil (U) which is found in RNA. The table has beenadapted so that frequencies are calculated for each amino acid, ratherthan for all 64 codons.

TABLE 2 Codon Usage Table for Saccharomyces cerevisiae Genes Frequencyper Amino Acid Codon Number hundred Phe UUU 170666 26.1 Phe UUC 12051018.4 Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Ile AUU 196893 30.1 IleAUC 112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU 14424322.1 Val GUC 76947 11.8 Val GUA 76927 11.8 Val GUG 70337 10.8 Ser UCU153557 23.5 Ser UCC 92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 SerAGU 92466 14.2 Ser AGC 63726 9.8 Pro CCU 88263 13.5 Pro CCC 44309 6.8Pro CCA 119641 18.3 Pro CCG 34597 5.3 Thr ACU 132522 20.3 Thr ACC 8320712.7 Thr ACA 116084 17.8 Thr ACG 52045 8.0 Ala GCU 138358 21.2 Ala GCC82357 12.6 Ala GCA 105910 16.2 Ala GCG 40358 6.2 Tyr UAU 122728 18.8 TyrUAC 96596 14.8 His CAU 89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3Gln CAG 79121 12.1 Asn AAU 233124 35.7 Asn AAC 162199 24.8 Lys AAA273618 41.9 Lys AAG 201361 30.8 Asp GAU 245641 37.6 Asp GAC 132048 20.2Glu GAA 297944 45.6 Glu GAG 125717 19.2 Cys UGU 52903 8.1 Cys UGC 310954.8 Trp UGG 67789 10.4 Arg CGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 195623.0 Arg CGG 11351 1.7 Arg AGA 139081 21.3 Arg AGG 60289 9.2 Gly GGU156109 23.9 Gly GGC 63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 StopUAA 6913 1.1 Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons optimal for a given species.Codon-optimized coding regions can be designed by various differentmethods.

In one method, a codon usage table is used to find the single mostfrequent codon used for any given amino acid, and that codon is usedeach time that particular amino acid appears in the polypeptidesequence. For example, referring to Table 2 above, for leucine, the mostfrequent codon is UUG, which is used 27.2% of the time. Thus all theleucine residues in a given amino acid sequence would be assigned thecodon UUG.

In another method, the actual frequencies of the codons are distributedrandomly throughout the coding sequence. Thus, using this method foroptimization, if a hypothetical polypeptide sequence had 100 leucineresidues, referring to Table 2 for frequency of usage in the S.cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11,or 11% of the leucine codons would be CUG, about 12, or 12% of theleucine codons would be CUU, about 13, or 13% of the leucine codonswould be CUA, about 26, or 26% of the leucine codons would be UUA, andabout 27, or 27% of the leucine codons would be UUG.

These frequencies would be distributed randomly throughout the leucinecodons in the coding region encoding the hypothetical polypeptide. Aswill be understood by those of ordinary skill in the art, thedistribution of codons in the sequence can vary significantly using thismethod; however, the sequence always encodes the same polypeptide.

When using the methods above, the term “about” is used precisely toaccount for fractional percentages of codon frequencies for a givenamino acid. As used herein, “about” is defined as one amino acid more orone amino acid less than the value given. The whole number value ofamino acids is rounded up if the fractional frequency of usage is 0.50or greater, and is rounded down if the fractional frequency of use is0.49 or less. Using again the example of the frequency of usage ofleucine in human genes for a hypothetical polypeptide having 62 leucineresidues, the fractional frequency of codon usage would be calculated bymultiplying 62 by the frequencies for the various codons. Thus, 7.28percent of 62 equals 4.51 UUA codons, or “about 5,” i.e., 4, 5, or 6 UUAcodons, 12.66 percent of 62 equals 7.85 UUG codons or “about 8,” i.e.,7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or“about 8,” i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percentof 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons,and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e.,24, 25, or 26 CUG codons.

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence, can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs are readily available to those of ordinaryskill in the art. For example, the “EditSeq” function in the LasergenePackage, available from DNAstar, Inc., Madison, Wis., thebacktranslation function in the VectorNTl Suite, available fromInforMax, Inc., Bethesda, Md., and the “backtranslate” function in theGCG—Wisconsin Package, available from Accelrys, Inc., San Diego, Calif.In addition, various resources are publicly available to codon-optimizecoding region sequences, e.g., the “backtranslation” function atwww.entelechon.com/2008/10/backtranslation-tool/ (visited Feb. 28, 2011)and the “backtranseq” function available atemboss.bioinformatics.nl/cgi-bin/emboss/backtranseq (visited Feb. 28,2011). Constructing a rudimentary algorithm to assign codons based on agiven frequency can also easily be accomplished with basic mathematicalfunctions by one of ordinary skill in the art.

A number of options are available for synthesizing codon optimizedcoding regions designed by any of the methods described above, usingstandard and routine molecular biological manipulations well known tothose of ordinary skill in the art. In one approach, a series ofcomplementary oligonucleotide pairs of 80-90 nucleotides each in lengthand spanning the length of the desired sequence is synthesized bystandard methods. These oligonucleotide pairs are synthesized such thatupon annealing, they form double stranded fragments of 80-90 base pairs,containing cohesive ends, e.g., each oligonucleotide in the pair issynthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond theregion that is complementary to the other oligonucleotide in the pair.The single-stranded ends of each pair of oligonucleotides is designed toanneal with the single-stranded end of another pair of oligonucleotides.The oligonucleotide pairs are allowed to anneal, and approximately fiveto six of these double-stranded fragments are then allowed to annealtogether via the cohesive single stranded ends, and then they ligatedtogether and cloned into a standard bacterial cloning vector, forexample, a TOPO® vector available from Invitrogen Corporation, Carlsbad,Calif. The construct is then sequenced by standard methods. Several ofthese constructs consisting of 5 to 6 fragments of 80 to 90 base pairfragments ligated together, i.e., fragments of about 500 base pairs, areprepared, such that the entire desired sequence is represented in aseries of plasmid constructs. The inserts of these plasmids are then cutwith appropriate restriction enzymes and ligated together to form thefinal construct. The final construct is then cloned into a standardbacterial cloning vector, and sequenced. Additional methods would beimmediately apparent to the skilled artisan. In addition, gene synthesisis readily available commercially.

In additional embodiments, a full-length polypeptide sequence iscodon-optimized for a given species resulting in a codon-optimizedcoding region encoding the entire polypeptide, and then nucleic acidfragments of the codon-optimized coding region, which encode fragments,variants, and derivatives of the polypeptide are made from the originalcodon-optimized coding region. As would be well understood by those ofordinary skill in the art, if codons have been randomly assigned to thefull-length coding region based on their frequency of use in a givenspecies, nucleic acid fragments encoding fragments, variants, andderivatives would not necessarily be fully codon optimized for the givenspecies. However, such sequences are still much closer to the codonusage of the desired species than the native codon usage. The advantageof this approach is that synthesizing codon-optimized nucleic acidfragments encoding each fragment, variant, and derivative of a givenpolypeptide, although routine, would be time consuming and would resultin significant expense.

Transposons

To select for foreign DNA that has entered a host it is preferable thatthe DNA be stably maintained in the organism of interest. With regard toplasmids, there are two processes by which this can occur. One isthrough the use of replicative plasmids. These plasmids have origins ofreplication that are recognized by the host and allow the plasmids toreplicate as stable, autonomous, extrachromosomal elements that arepartitioned during cell division into daughter cells. The second processoccurs through the integration of a plasmid onto the chromosome. Thispredominately happens by homologous recombination and results in theinsertion of the entire plasmid, or parts of the plasmid, into the hostchromosome. Thus, the plasmid and selectable marker(s) are replicated asan integral piece of the chromosome and segregated into daughter cells.Therefore, to ascertain if plasmid DNA is entering a cell during atransformation event through the use of selectable markers requires theuse of a replicative plasmid or the ability to recombine the plasmidonto the chromosome. These qualifiers cannot always be met, especiallywhen handling organisms that do not have a suite of genetic tools.

One way to avoid issues regarding plasmid-associated markers is throughthe use of transposons. A transposon is a mobile DNA element, defined bymosaic DNA sequences that are recognized by enzymatic machinery referredto as a transposase. The function of the transposase is to randomlyinsert the transposon DNA into host or target DNA. A selectable markercan be cloned onto a transposon by standard genetic engineering. Theresulting DNA fragment can be coupled to the transposase machinery in anin vitro reaction and the complex can be introduced into target cells byelectroporation. Stable insertion of the marker onto the chromosomerequires only the function of the transposase machinery and alleviatesthe need for homologous recombination or replicative plasmids.

The random nature associated with the integration of transposons has theadded advantage of acting as a form of mutagenesis. Libraries can becreated that comprise amalgamations of transposon mutants. Theselibraries can be used in screens or selections to produce mutants withdesired phenotypes. For instance, a transposon library of a CBP organismcould be screened for the ability to produce more ethanol, or lesslactic acid and/or more acetate.

Native Cellulolytic Strategy

Naturally occurring cellulolytic microorganisms are starting points forCBP organism development via the native strategy. Anaerobes andfacultative anaerobes are of particular interest. The primary objectiveis to engineer the metabolization of biomass to solvents, including butnot limited to, acetone, isopropanol, ethyl acetate, or ethanol.Metabolic engineering of mixed-acid fermentations in relation to, forexample, ethanol production, has been successful in the case ofmesophilic, non-cellulolytic, enteric bacteria. Recent developments insuitable gene-transfer techniques allow for this type of work to beundertaken with cellulolytic bacteria.

Recombinant Cellulolytic Strategy

Non-cellulolytic microorganisms with desired product-formationproperties are starting points for CBP organism development by therecombinant cellulolytic strategy. The primary objective of suchdevelopments is to engineer a heterologous cellulase system that enablesgrowth and fermentation on pretreated lignocellulose. The heterologousproduction of cellulases has been pursued primarily with bacterial hostsproducing ethanol at high yield (engineered strains of E. coli,Klebsiella oxytoca, and Zymomonas mobilis) and the yeast Saccharomycescerevisiae. Cellulase expression in strains of K. oxytoca resulted inincreased hydrolysis yields—but not growth without added cellulase—formicrocrystalline cellulose, and anaerobic growth on amorphous cellulose.Although dozens of saccharolytic enzymes have been functionallyexpressed in S. cerevisiae, anaerobic growth on cellulose as the resultof such expression has not been definitively demonstrated.

Aspects of the present invention relate to the use of thermophilic ormesophilic microorganisms as hosts for modification via the nativecellulolytic strategy. Their potential in process applications inbiotechnology stems from their ability to grow at relatively hightemperatures with attendant high metabolic rates, production ofphysically and chemically stable enzymes, and elevated yields of endproducts. Major groups of thermophilic bacteria include eubacteria andarchaebacteria. Thermophilic eubacteria include: phototropic bacteria,such as cyanobacteria, purple bacteria, and green bacteria;Gram-positive bacteria, such as Bacillus, Clostridium, Lactic acidbacteria, and Actinomyces; and other eubacteria, such as Thiobacillus,Spirochete, Desulfotomaculum, Gram-negative aerobes, Gram-negativeanaerobes, and Thermotoga. Within archaebacteria are consideredMethanogens, extreme thermophiles (an art-recognized term), andThermoplasma. In certain embodiments, the present invention relates toGram-negative organotrophic thermophiles of the genera Thermus,Gram-positive eubacteria, such as genera Clostridium, and also whichcomprise both rods and cocci, genera in group of eubacteria, such asThermosipho and Thermotoga, genera of Archaebacteria, such asThermococcus, Thermoproteus (rod-shaped), Thermofilum (rod-shaped),Pyrodictium, Acidianus, Sulfolobus, Pyrobaculum, Pyrococcus,Thermodiscus, Staphylothermus, Desulfurococcus, Archaeoglobus, andMethanopyrus. Some examples of thermophilic or mesophilic (includingbacteria, procaryotic microorganism, and fungi), which may be suitablefor the present invention include, but are not limited to: Clostridiumthermosulfurogenes, Clostridium cellulolyticum, Clostridiumthermocellum, Clostridium the rmohydrosulfuricum, Clostridiumthermoaceticum, Clostridium thermosaccharolyticum, Clostridiumtartarivorum, Clostridium thermocellulaseum, Clostridiumphytofermentans, Clostridium straminosolvens, Thermoanaerobacteriumthermosaccarolyticum, Thermoanaerobacterium saccharolyticum,Thermobacteroides acetoethylicus, Thermoanaerobium brockii,Methanobacterium thermoautotrophicum, Anaerocellum thermophilium,Pyrodictium occultum, Thermoproteus neutrophilus, Thermofilum librum,Thermothrix thioparus, Desulfovibrio thermophilus, Thermoplasmaacidophilum, Hydrogenomonas thermophilus, Thermomicrobium roseum,Thermus flavas, Thermus ruber, Pyrococcus furiosus, Thermus aquaticus,Thermus thermophilus, Chloroflexus aurantiacus, Thermococcus litoralis,Pyrodictium abyssi, Bacillus stearothermophilus, Cyanidium caldarium,Mastigocladus laminosus, Chlamydothrix calidissima, Chlamydothrixpenicillata, Thiothrix carnea, Phormidium tenuissimum, Phormidiumgeysericola, Phormidium subterraneum, Phormidium bijahensi, Oscillatoriafiliformis, Synechococcus lividus, Chloroflexus aurantiacus, Pyrodictiumbrockii, Thiobacillus thiooxidans, Sulfolobus acidocaldarius,Thiobacillus thermophilica, Bacillus stearothermophilus, Cercosulciferhamathensis, Vahlkampfia reichi, Cyclidium citrullus, Dactylariagallopava, Synechococcus lividus, Synechococcus elongatus, Synechococcusminervae, Synechocystis aquatilus, Aphanocapsa thermalis, Oscillatoriaterebriformis, Oscillatoria amphibia, Oscillatoria germinate,Oscillatoria okenii, Phormidium laminosum, Phormidium parparasiens,Symploca thermalis, Bacillus acidocaldarias, Bacillus coagulans,Bacillus thermocatenalatus, Bacillus licheniformis, Bacillus pamilas,Bacillus macerans, Bacillus circulans, Bacillus laterosporus, Bacillusbrevis, Bacillus subtilis, Bacillus sphaericus, Desulfotomaculumnigrificans, Streptococcus thermophilus, Lactobacillus thermophilus,Lactobacillus bulgaricus, Bifidobacterium thermophilum, Streptomycesfragmentosporus, Streptomyces thermonitrificans, Streptomycesthermovulgaris, Pseudonocardia thermophila, Thermoactinomyces vulgaris,Thermoactinomyces sacchari, Thermoactinomyces candidas, Thermomonosporacurvata, Thermomonospora viridis, Thermomonospora citrina, Microbisporathermodiastatica, Microbispora aerata, Microbispora bispora,Actinobifida dichotomica, Actinobifida chromogens, Micropolysporacaesia, Micropolyspora faeni, Micropolyspora cectivugida, Micropolysporacabrobrunea, Micropolyspora thermovirida, Micropolyspora viridinigra,Methanobacterium thermoautothropicum, Caldicellulosiruptor acetigenus,Caldicellulosiruptor saccharolyticus, Caldicellulosiruptorkristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptorlactoaceticus, variants thereof, and/or progeny thereof.

In particular embodiments, the present invention relates to thermophilicbacteria selected from the group consisting of Clostridiumcellulolyticum, Clostridium thermocellum, and Thermoanaerobacteriumsaccharolyticum.

In certain embodiments, the present invention relates to thermophilicbacteria selected from the group consisting of Fervidobacteriumgondwanense, Clostridium thermolacticum, Moorella sp., and Rhodothermusmarinus.

In certain embodiments, the present invention relates to thermophilicbacteria of the genera Thermoanaerobacterium or Thermoanaerobacter,including, but not limited to, species selected from the groupconsisting of: Thermoanaerobacterium thermosulfurigenes,Thermoanaerobacterium aotearoense, Thermoanaerobacteriumpolysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacteriumxylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobiumbrockii, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterthermohydrosulfuricus, Thermoanaerobacter ethanolicus,Thermoanaerobacter brockii, variants thereof, and progeny thereof.

In certain embodiments, the present invention relates to microorganismsof the genera Geobacillus, Saccharococcus, Paenibacillus, Bacillus, andAnoxybacillus, including, but not limited to, species selected from thegroup consisting of: Geobacillus thermoglucosidasius, Geobacillusstearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccusthermophilus, Paenibacillus campinasensis, Bacillus flavothermus,Anoxybacillus kamchatkensis, Anoxybacillus gonensis, variants thereof,and progeny thereof.

In certain embodiments, the present invention relates to mesophilicbacteria selected from the group consisting of Saccharophagus degradans;Flavobacterium johnsoniae; Fibrobacter succinogenes; Clostridiumhungatei; Clostridium phytofermentans; Clostridium cellulolyticum;Clostridium aldrichii; Clostridium termitididis; Acetivibriocellulolyticus; Acetivibrio ethanolgignens; Acetivibrio multivorans;Bacteroides cellulosolvens; and Alkalibacter saccharofomentans, variantsthereof and progeny thereof.

Organism Development Via the Native Cellulolytic Strategy

One approach to organism development for CBP begins with organisms thatnaturally utilize cellulose, hemicellulose and/or other biomasscomponents, which are then genetically engineered to enhance productyield and tolerance. For example, Clostridium thermocellum is athermophilic bacterium that has among the highest rates of celluloseutilization reported. Other organisms of interest are xylose-utilizingthermophiles such as Thermoanaerobacterium saccharolyticum andThermoanaerobacterium thermosaccharolyticum. Organic acid production maybe responsible for the low concentrations of produced ethanol generallyassociated with these organisms. Thus, one objective is to eliminateproduction of acetic and lactic acid in these organisms via metabolicengineering. Substantial efforts have been devoted to developing genetransfer systems for the above-described target organisms and multipleC. thermocellum isolates from nature have been characterized. SeeMcLaughlin et al. (2002) Environ. Sci. Technol. 36:2122. Metabolicengineering of thermophilic, saccharolytic bacteria is an active area ofinterest, and knockout of lactate dehydrogenase in T. saccharolyticumhas recently been reported. See Desai et al. (2004) Appl. Microbiol.Biotechnol. 65:600. Knockout of acetate kinase and phosphotransacetylasein this organism is also possible.

Organism Development Via the Recombinant Cellulolytic Strategy

An alternative approach to organism development for CBP involvesconferring the ability to grow on lignocellulosic materials tomicroorganisms that naturally have high product yield and tolerance viaexpression of a heterologous cellulasic system and perhaps otherfeatures. For example, Saccharomyces cerevisiae has been engineered toexpress over two dozen different saccharolytic enzymes. See Lynd et al.(2002) Microbiol. Mol. Biol. Rev. 66:506.

Whereas cellulosic hydrolysis has been approached in the literatureprimarily in the context of an enzymatically-oriented intellectualparadigm, the CBP processing strategy requires that cellulosichydrolysis be viewed in terms of a microbial paradigm. This microbialparadigm naturally leads to an emphasis on different fundamental issues,organisms, cellulasic systems, and applied milestones compared to thoseof the enzymatic paradigm. In this context, C. thermocellum has been amodel organism because of its high growth rate on cellulose togetherwith its potential utility for CBP.

In certain embodiments, organisms useful in the present invention may beapplicable to the process known as simultaneous saccharification andfermentation (SSF), which is intended to include the use of saidmicroorganisms and/or one or more recombinant hosts (or extractsthereof, including purified or unpurified extracts) for thecontemporaneous degradation or depolymerization of a complex sugar(i.e., cellulosic biomass) and bioconversion of that sugar residue intoethanol by fermentation.

Ethanol Production

According to the present invention, a recombinant microorganism can beused to produce ethanol from biomass, which is referred to herein aslignocellulosic material, lignocellulosic substrate, or cellulosicbiomass. Methods of producing ethanol can be accomplished, for example,by contacting the biomass with a recombinant microorganism as describedherein, and as described in commonly owned U.S. Patent ApplicationPublication No. 2011/0189744 A1, U.S. Patent Application Publication No.2011/0312054 A1, U.S. Patent Application Publication No. 2012/0003701,International Appl. No. PCT/US2009/065571, International Appl. No.PCT/US2009/069443, International Appl. No. PCT/US2009/064128,International Appl. No. PCT/IB2009/005881, and PCT/US2009/065571, thecontents of each are incorporated by reference herein.

Numerous cellulosic substrates can be used in accordance with thepresent invention. Substrates for cellulose activity assays can bedivided into two categories, soluble and insoluble, based on theirsolubility in water. Soluble substrates include cellodextrins orderivatives, carboxymethyl cellulose (CMC), or hydroxyethyl cellulose(HEC). Insoluble substrates include crystalline cellulose,microcrystalline cellulose (Avicel), amorphous cellulose, such asphosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose,and pretreated lignocellulosic biomass. These substrates are generallyhighly ordered cellulosic material and thus only sparingly soluble.

It will be appreciated that suitable lignocellulosic material may be anyfeedstock that contains soluble and/or insoluble cellulose, where theinsoluble cellulose may be in a crystalline or non-crystalline form. Invarious embodiments, the lignocellulosic biomass comprises, for example,wood, corn, corn stover, sawdust, bark, leaves, agricultural andforestry residues, grasses such as switchgrass, ruminant digestionproducts, municipal wastes, paper mill effluent, newspaper, cardboard orcombinations thereof.

In some embodiments, the invention is directed to a method forhydrolyzing a cellulosic substrate, for example a cellulosic substrateas described above, by contacting the cellulosic substrate with arecombinant microorganism of the invention. In some embodiments, theinvention is directed to a method for hydrolyzing a cellulosicsubstrate, for example a cellulosic substrate as described above, bycontacting the cellulosic substrate with a co-culture comprising yeastcells expressing heterologous cellulases.

In some embodiments, the invention is directed to a method forfermenting cellulose. Such methods can be accomplished, for example, byculturing a host cell or co-culture in a medium that contains insolublecellulose to allow saccharification and fermentation of the cellulose.

The production of ethanol can, according to the present invention, beperformed at temperatures of at least about 30° C., about 31° C., about32° C., about 33° C., about 34° C., about 35° C., about 36° C., about37° C., about 38° C., about 39° C., about 40° C., about 41° C., about42° C., about 43° C., about 44° C., about 45° C., about 46° C., about47° C., about 48° C., about 49° C., or about 50° C. In some embodimentsof the present invention the thermotolerant host cell can produceethanol from cellulose at temperatures above about 30° C., about 31° C.,about 32° C., about 33° C., about 34° C., about 35° C., about 36° C.,about 37° C., about 38° C., about 39° C., about 40° C., about 41° C.,about 42° C., or about 43° C., or about 44° C., or about 45° C., orabout 50° C. In some embodiments of the present invention, thethermotolterant host cell can produce ethanol from cellulose attemperatures from about 30° C. to 60° C., about 30° C. to 55° C., about30° C. to 50° C., about 40° C. to 60° C., about 40° C. to 55° C. orabout 40° C. to 50° C.

In some embodiments, methods of producing ethanol can comprisecontacting a cellulosic substrate with a recombinant microorganism orco-culture of the invention and additionally contacting the cellulosicsubstrate with externally produced cellulase enzymes. Exemplaryexternally produced cellulase enzymes are commercially available and areknown to those of skill in the art.

In some embodiments, the methods comprise producing ethanol at aparticular rate. For example, in some embodiments, ethanol is producedat a rate of at least about 0.1 mg per hour per liter, at least about0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, atleast about 0.75 mg per hour per liter, at least about 1.0 mg per hourper liter, at least about 2.0 mg per hour per liter, at least about 5.0mg per hour per liter, at least about 10 mg per hour per liter, at leastabout 15 mg per hour per liter, at least about 20.0 mg per hour perliter, at least about 25 mg per hour per liter, at least about 30 mg perhour per liter, at least about 50 mg per hour per liter, at least about100 mg per hour per liter, at least about 200 mg per hour per liter, atleast about 300 mg per hour per liter, at least about 400 mg per hourper liter, or at least about 500 mg per hour per liter.

In some embodiments, the host cells of the present invention can produceethanol at a rate of at least about 0.1 mg per hour per liter, at leastabout 0.25 mg per hour per liter, at least about 0.5 mg per hour perliter, at least about 0.75 mg per hour per liter, at least about 1.0 mgper hour per liter, at least about 2.0 mg per hour per liter, at leastabout 5.0 mg per hour per liter, at least about 10 mg per hour perliter, at least about 15 mg per hour per liter, at least about 20.0 mgper hour per liter, at least about 25 mg per hour per liter, at leastabout 30 mg per hour per liter, at least about 50 mg per hour per liter,at least about 100 mg per hour per liter, at least about 200 mg per hourper liter, at least about 300 mg per hour per liter, at least about 400mg per hour per liter, or at least about 500 mg per hour per liter morethan a control strain (e.g., a wild-type strain) and grown under thesame conditions. In some embodiments, the ethanol can be produced in theabsence of any externally added cellulases.

Ethanol production can be measured using any method known in the art.For example, the quantity of ethanol in fermentation samples can beassessed using HPLC analysis. Many ethanol assay kits are commerciallyavailable that use, for example, alcohol oxidase enzyme based assays.Methods of determining ethanol production are within the scope of thoseskilled in the art from the teachings herein. The U.S. Department ofEnergy (DOE) provides a method for calculating theoretical ethanolyield. Accordingly, if the weight percentages are known of C6 sugars(i.e., glucan, galactan, mannan), the theoretical yield of ethanol ingallons per dry ton of total C6 polymers can be determined by applying aconversion factor as follows:(1.11 pounds of C6 sugar/pound of polymeric sugar)×(0.51 pounds ofethanol/pound of sugar)×(2000 pounds of ethanol/ton of C6 polymericsugar)×(1 gallon of ethanol/6.55 pounds of ethanol)×( 1/100%), whereinthe factor (1 gallon of ethanol/6.55 pounds of ethanol) is taken as thespecific gravity of ethanol at 20° C.

And if the weight percentages are known of C5 sugars (i.e., xylan,arabinan), the theoretical yield of ethanol in gallons per dry ton oftotal C5 polymers can be determined by applying a conversion factor asfollows:(1.136 pounds of C5 sugar/pound of C5 polymeric sugar)×(0.51 pounds ofethanol/pound of sugar)×(2000 pounds of ethanol/ton of C5 polymericsugar)×(1 gallon of ethanol/6.55 pounds of ethanol)×( 1/100%), whereinthe factor (1 gallon of ethanol/6.55 pounds of ethanol) is taken as thespecific gravity of ethanol at 20° C.

It follows that by adding the theoretical yield of ethanol in gallonsper dry ton of the total C6 polymers to the theoretical yield of ethanolin gallons per dry ton of the total C5 polymers gives the totaltheoretical yield of ethanol in gallons per dry ton of feedstock.

Applying this analysis, the DOE provides the following examples oftheoretical yield of ethanol in gallons per dry ton of feedstock: corngrain, 124.4; corn stover, 113.0; rice straw, 109.9; cotton gin trash,56.8; forest thinnings, 81.5; harwood sawdust, 100.8; bagasse, 111.5;and mixed paper, 116.2. It is important to note that these aretheoretical yields. The DOE warns that depending on the nature of thefeedstock and the process employed, actual yield could be anywhere from60% to 90% of theoretical, and further states that “achieving high yieldmay be costly, however, so lower yield processes may often be more costeffective.” (Ibid.)

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Improving Ethanol Yield Through Engineering of AlternateElectron Acceptors

The present Example describes pathways to reduce or eliminate glycerolby engineering alternate electron acceptors in a yeast cell. Glycerol isan undesired by-product of sugar metabolism during anaerobic growth inyeast. The amount of glycerol produced during anaeroblic growth onglucose has been empirically determined by Medina, V G, et al., Appl.Env. Microbiol. 76:190-95 (2010):56 mmol glucose→1 g biomass+88 mmol ethanol+95 mmol CO₂+11 mmolglycerol+1.7 mmol acetate

Assuming glycerol production is primarily for the regeneration of NAD⁺for the continuation of glycolysis, a half reaction for glycerolproduction is (Medina, V G, et al., Appl. Env. Microbiol. 76:190-95(2010)):0.5 glucose+NADH+H⁺+ATP→glycerol+NAD⁺ADP+P_(i)

The following pathways describe engineering an alternate electronacceptor for glycerol in the above half reaction, engineering anincrease in ethanol yield during anaerobic growth on glucose by usingimproved enzyme activities for converting glucose to ethanol, and/ordeleting endogenous glycerol-producing or glycerol-regulating genes.

1.1 Engineering of a Formate Pathway in Yeast

Production of formate from glucose can provide similar reducingequivalents as glycerol, as shown in the following half reaction:0.5 glucose+NADH+H⁺+ADP+P_(i)→formate+NAD⁺+ATP+ethanol

In addition to balancing the redox constraints of the cell, this pathwayprovides increased ATP yield and results in an overall anaerobic growthequation of:56 mmol glucose→1 g biomass+99 mmol ethanol+95 mmol CO₂+11 mmolformate+1.7 mmol acetate

Engineering in a formate pathway as an alternate electron acceptor toglycerol results in an increase of 12.5% in the theoretical yield ofethanol. Enzymes than can be targeted to engineer such an increaseinclude pyruvate formate lyase (PFL) and formate dehydrogenase (FDH).See FIG. 1.

1.1.1 Expression of PFL

The conversion of pyruvate to acetyl-CoA and formate is performed byPFL.

Thus, to produce formate in yeast, a PFL can be expressed. PFLs arecommon in bacteria from a variety of sources. Vigorous hydrogenproducing bacteria, such as from a clostridium, thermoanaerobacterium,or other anaerobic bacteria will likely result in an increasedproductivity. Examples of PFL include, but are not limited, Bacilluslicheniformis, Streptococcus thermophilus, Lactobacillus plantarum,Lactobacillus casei, Bifidobacterium adolescentis, Clostridiumcellulolyticum, Escherichia coli, Chlamydomonas reinhardtii PfIA,Piromyces sp. E2, or Neocallimastix frontalis. See Example 4 and Table 1below.

1.1.2 Deletion of FDH

To prevent yeast from converting formate to CO₂ and NADH, endogeneousFDH genes can be deleted or downregulated. Deleting or downregulatingfdh1, fdh2, or both genes can enhance the redox balance and ethanolyield of the recombinant microorganisms of the invention.

1.2 Improving Conversion of Acetyl-CoA to Ethanol

To improve the conversion of acetyl-CoA to ethanol, endogenous yeastgenes can be replaced or complimented with either an improvedacetaldehyde dehydrogenase (e.g., from C. phytofermentans or othersource) to convert acetyl-CoA to acetaldehyde, or a bifunctionalacetaldehyde/alcohol dehydrogenase (AADH) to convert acetyl-CoA toacetaldehyde and acetaldehyde to ethanol. By engineering in one or moresuch enzymes, the in vivo kinetics of the conversion of acetyl-CoA toethanol can be increased, providing for improved growth of the hoststrain. The bi-functional alcohol/aldehyde dehydrogenase can come from avariety of microbial sources, including but not limited to E. coli, C.acetobutylicum, T saccharolyticum, C. thermocellum, C. phytofermentans,Piromyces SP E2, or Bifidobacterium adolescentis.

1.3 Deletion or Downregulation of Glycerol Pathway

Deleting or altering expression of glycerol formation genes will reduceor block endogenous production of glycerol and may enhance acetateuptake. Deletion of gpd1, gpd2, or both genes and/or deletion of gpp1,gpp2, or both genes may be used to eliminate glycerol formation andenhance ethanol yield. However, the complete elimination of glycerol maynot be practical for an industrial process. See Guo, Z P., et al.,Metab. Eng. 13:49-59 (2011). Thus, rather than the complete removal ofany one, all, or some combination of these glycerol formation genes, oneor more of these genes can be altered or downregulated to reduceglycerol formation and enhance ethanol yield.

Example 2 Deletion or Downregulation of Glycerol-Regulating Gene FPS 1to Improve Ethanol Yield

Instead of, or in addition to, downregulating glycerol productionthrough deletion or alteration of glycerol-forming genes, glycerolproduction can be downregulated by deletion or alteration of aglycerol-regulating gene. FPS1 is an aquaglyceroporin responsible forefflux of glycerol. An fps1Δ strain has reduced glycerol formation yethas a completely functional NAD⁺-dependant glycerol synthesis pathway.In addition to deletion of FPS1, constitutively active mutants of FPS1or homologs from other organisms can be expressed to alter glycerolproduction. Because such FPS1 deletion or alteration strains can stillsynthesize and retain glycerol, improved robustness may be observedrelative to strains that are unable to make glycerol.

Null mutants of an fps1Δ strain grow much slower anaerobically thanwild-type due to intracellular glycerol accumulation. Tamás, M. J., etal., Molecular Microbiol. 31(4):1087-1104 (1999). However, preliminarydata indicates that expression of a B. adolescentis bifunctional AADH inconjunction with B. adolescentis PFL in an fps1Δ strain can enableanaerobic growth of fps1Δ strain (see Example 7 and FIGS. 22 and 24).Additionally, significantly improved osmotic tolerance was also observedwhen FPS1 was deleted in glycerol mutant strains containing ADH and PFLalone. Increased resistance to osmotic stress was determined byobservation of improved growth of the fps1Δ mutant on several differenthigh osmolarity media including 1M sodium chloride, 1M sorbitol, and 1Mxylitol. The fps1Δ mutant was created by marker recycle resulting in adeletion of a large region of the FPS1 coding sequence (sequence ofnative and deletion below).

Sequence of FPS1 locus (coding sequence is underlined; SEQ ID NO:104):

aacgcggctgatgcttttatttaggaaggaatacttacattatcatgagaacattgtcaagggcattctgatacgggccttccatcgcaagaaaaaggcagcaacggactgagggacggagagagttacggcataagaagtagtaggagagcagagtgtcataaagttatattattctcgtcctaaagtcaattagttctgttgcgcttgacaatatatgtcgtgtaataccgtcccttagcagaagaaagaaagacggatccatatatgttaaaatgcttcagagatgtttctttaatgtgccgtccaacaaaggtatcttctgtagcttcctctattttcgatcagatctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtgaatccggagacggcaagattgcccggccctttttgcggaaaagataaaacaagatatattgcactttttccaccaagaaaaacaggaagtggattaaaaaatcaacaaagtataacgcctattgtcccaataagcgtcggttgttcttctttattattttaccaagtacgctcgagggtacattctaatgcattaaaagacatgagtaatcctcaaaaagctctaaacgactttctgtccagtgaatctgttcatacacatgatagttctaggaaacaatctaataagcagtcatccgacgaaggacgctcttcatcacaaccttcacatcatcactctggtggtactaacaacaataataacaataataataataataataacagtaacaacaacaacaacggcaacgatgggggaaatgatgacgactatgattatgaaatgcaagattatagaccttctccgcaaagtgcgcggcctactcccacgtatgttccacaatattctgtagaaagtgggactgctttcccgattcaagaggttattcctagcgcatacattaacacacaagatataaaccataaagataacggtccgccgagtgcaagcagtaatagagcattcaggcctagagggcagaccacagtgtcggccaacgtgcttaacattgaagatttttacaaaaatgcagacgatgcgcataccatcccggagtcacatttatcgagaaggagaagtaggtcgagggctacgagtaatgctgggcacagtgccaatacaggcgccacgaatggcaggactactggtgcccaaactaatatggaaagcaatgaatcaccacgtaacgtccccattatggtgaagccaaagacattataccagaaccctcaaacacctacagtcttgccctccacataccatccaattaataaatggtcttccgtcaaaaacacttatttgaaggaatttttagccgagtttatgggaacaatggttatgattattttcggtagtgctgttgtttgtcaggtcaatgttgctgggaaaatacagcaggacaatttcaacgtggctttggataaccttaacgttaccgggtcttctgcagaaacgatagacgctatgaagagtttaacatccttggtttcatccgttgcgggcggtacctttgatgatgtggcattgggctgggctgctgccgtggtgatgggctatttctgcgctggtggtagtgccatctcaggtgctcatttgaatccgtctattacattagccaatttggtgtatagaggttttcccctgaagaaagttccttattactttgctggacaattgatcggtgccttcacaggcgctttgatcttgtttatttggtacaaaagggtgttacaagaggcatatagcgattggtggatgaatgaaagtgttgcgggaatgttttgcgtttttccaaagccttatctaagttcaggacggcaatttttttccgaatttttatgtggagctatgttacaagcaggaacatttgcgctgaccgatccttatacgtgtttgtcctctgatgttttcccattgatgatgtttattttgattttcattatcaatgcttccatggcttatcagacaggtacagcaatgaatttggctcgtgatctgggcccacgtcttgcactatatgcagttggatttgatcataaaatgctttgggtgcatcatcatcatttcttttgggttcccatggtaggcccatttattggtgcgttaatgggggggttggtttacgatgtctgtatttatcagggtcatgaatctccagtcaactggtctttaccagtttataaggaaatgattatgagagcctggtttagaaggcctggttggaagaagagaaatagagcaagaagaacatcggacctgagtgacttctcatacaataacgatgatgatgaggaatttggagaaagaatggctcttcaaaagacaaagaccaagtcatctatttcagacaacgaaaatgaagcaggagaaaagaaagtgcaatttaaatctgttcagcgcggcaaaagaacgtttggtggtataccaacaattcttgaagaagaagattccattgaaactgcttcgctaggtgcgacgacgactgattctattgggttatccgacacatcatcagaagattcgcattatggtaatgctaagaaggtaacatgagaaaacagacaagaaaaagaaacaaataatatagactgatagaaaaaaatactgcttactaccgccggtataatatatatatatatatatatttacatagatgattgcatagtgttttaaaaagctttcctaggttaagctatgaatcttcataacctaaccaactaaatatgaaaatactgacccatcgtcttaagtaagttgacatgaactcagcctggtcacctactatacatgatgtatcgcatggatggaaagaataccaaacgctaccttccaggttaatgatagtatccaaacctagttggaatttgccttgaacatcaagcagcgattcgatatcagttgggagcatcaatttggtcattggaataccatctatgcttttctcctcccatattcgcaaaagtagtaagggctcgttatatacttttgaatatgtaagatataattctatatgatttagtaatttattttctatacgctcagtatttttctgcagttgtcgagtaggtattaaacgcaaaagaagtccatccttttcatcattcaaatggacatcttggcaaagggcccagttatggaaaatctgggagtcatacaacgattgcagttggctatgccactcctggtaaggaatcatcaagtctgataattctgttttttagccctttttttttttttttcatg

Sequence of fps1Δ mutation (part of the fps1 coding sequence was notdeleted (underlined) and the region that was deleted is represented by aΔ; SEQ ID NO:105):

aacgcggctgatgcttttatttaggaaggaatacttacattatcatgagaacattgtcaagggcattctgatacgggccttccatcgcaagaaaaaggcagcaacggactgagggacggagagagttacggcataagaagtagtaggagagcagagtgtcataaagttatattattctcgtcctaaagtcaattagttctgttgcgcttgacaatatatgtcgtgtaataccgtcccttagcagaagaaagaaagacggatccatatatgttaaaatgcttcagagatgtttctttaatgtgccgtccaacaaaggtatcttctgtagcttcctctattttcgatcagatctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtgaatccggagacggcaagattgcccggccctttttgcggaaaagataaaacaagatatattgcactttttccaccaagaaaaacaggaagtggattaaaaaatcaacaaagtataacgcctattgtcccaataagcgtcggttgttcttctttattattttaccaagtacgctcgagggtacattctaatgcattaaaagacΔgattcgcattatggtaatgctaagaaggtaacatgagaaaacagacaagaaaaagaaacaaataatatagactgatagaaaaaaatactgcttactaccgccggtataatatatatatatatatatatttacatagatgattgcatagtgttttaaaaagctttcctaggttaagctatgaatcttcataacctaaccaactaaatatgaaaatactgacccatcgtcttaagtaagttgacatgaactcagcctggtcacctactatacatgatgtatcgcatggatggaaagaataccaaacgctaccttccaggttaatgatagtatccaaacctagttggaatttgccttgaacatcaagcagcgattcgatatcagttgggagcatcaatttggtcattggaataccatctatgcttttctcctcccatattcgcaaaagtagtaagggctcgttatatacttttgaatatgtaagatataattctatatgatttagtaatttattttctatacgctcagtatttttctgcagttgtcgagtaggtattaaacgcaaaagaagtccatccttttcatcattcaaatggacatcttggcaaagggcccagttatggaaaatctgggagtcatacaacgattgcagttggctatgccactcctggtaaggaatcatcaagtctgataattctgttttttagccctttttttttttttttcatg

Example 3 Generating Yeast Strains with a Deleted or DownregulatedGlycerol-Production Pathway

To create yeast strains with altered glycerol production, endogenousglycerol-producing or regulating genes can either be deleted ordownregulated, by generating the following genetic backgrounds:

Haploid Strains Diploid Strains Glycerol gpd1Δ gpd2Δ fdh1Δ fdh2Δgpd1Δ/gpd1Δ gpd2Δ/gpd2Δ Elimination fdh1Δ fdh2Δ fps1Δ fdh1Δ/fdh1Δfdh2Δ/fdh2Δ Background gpd1Δ gpd2Δ fdh1Δ fdh2Δ fdh1Δ/fdh1Δ fdh2Δ/fdh2Δfps1Δ fps1Δ/fps1Δ gpd1Δ gpd2Δ fps1Δ gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ gpd1Δ gpd2Δfdh1Δ/fdh1Δ fdh2Δ/fdh2Δ fps1Δ/fps1Δ gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ fps1Δ/fps1Δgpd1Δ/gpd1Δ gpd2Δ/gpd2Δ Glycerol gpd1Δ gpd2Δ::GPD1 fdh1Δ gpd1Δ/gpd1ΔReduction fdh2Δ gpd2Δ/gpd2Δ::GPD1/GPD1 Background gpd1Δ gpd2Δ::GPD1fdh1Δ/fdh1Δ fdh2Δ/fdh2Δ gpd1Δ gpd2Δ::GPD1 gpd1Δ/gpd1Δ fdh1Δfdh2Δ fps1Δgpd2Δ/gpd2Δ::GPD1/GPD1 gpd1Δ gpd2Δ::GPD1 fps1Δ gpd1Δ/gpd1Δ gpd1Δ: GPD2gpd2Δ gpd2Δ/gpd2Δ::GPD1/GPD1 GPD1::GPD2 gpd2Δ fdh1Δ/fdh1Δ fdh2Δ/fdh2Δfdh1Δfdh2Δ fps1Δ/fps1Δ gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ::GPD1/GPD1 fps1Δ/fps1Δgpd1Δ/gpd1Δ::GPD2/GPD2 gpd2Δ/gpd2Δ gpd1Δ/gpd1Δ::GPD2/GPD2 gpd2Δ/gpd2Δfdh1Δ/fdh1Δ fdh2Δ/fdh2Δ

Strains in the glycerol elimination background were created by deletingone or more of the following genes: gpd1, gpd2, fdh1, fdh2, and/or fps1.Strains in the glycerol reduction background have been created by bydeleting one or more of the following genes: gpd1, gpd2, fdh1, fdh2,and/or fps1, and by expressing GPD 1 under the control of the gpd2promoter (designated gpd2Δ:: GPD1). These strains in which GPD1 isexpressed from the gpd2 promoter make a smaller amount of glycerolrelative to a wild-type strain.

3.1 Generation of Glycerol-Elimination Strain gpd1Δ gpd2Δ fdh1Δ fdh2Δ

To produce glycerol-elimination strain gpd1Δ gpd2Δ fdh1Δ fdh2Δ, thefollowing methods were used. All genetic modications were generatedusing positive selections to insert genetic elements and negativeselections to remove genetic elements. See FIGS. 4-11. The geneticelements were amplified by PCR and transformed into host strains,followed by selection and screening for the desired modification. Thesequence of native gpd1, gpd2, fdh1, fdh2 from S. cerevisiae, andresulting loci following deletion, are listed below.

Sequence of GPD1 locus (coding sequence is underlined; SEQ ID NO:89):

tacaaacgcaacacgaaagaacaaaaaaagaagaaaacagaaggccaagacagggtcaatgagactgttgtcctcctactgtccctatgtctctggccgatcacgcgccattgtccctcagaaacaaatcaaacacccacaccccgggcacccaaagtccccacccacaccaccaatacgtaaacggggcgccccctgcaggccctcctgcgcgcggcctcccgccttgcttctctccccttccttttctttttccagttttccctattttgtccctttttccgcacaacaagtatcagaatgggttcatcaaatctatccaacctaattcgcacgtagactggcttggtattggcagtttcgtagttatatatatactaccatgagtgaaactgttacgttaccttaaattctttctccctttaattttcttttatcttactctcctacataagacatcaagaaacaattgtatattgtacaccccccccctccacaaacacaaatattgataatataaagatgtctgctgctgctgatagattaaacttaacttccggccacttgaatgctggtagaaagagaagttcctcttctgtttctttgaaggctgccgaaaagcctttcaaggttactgtgattggatctggtaactggggtactactattgccaaggtggttgccgaaaattgtaagggatacccagaagttttcgctccaatagtacaaatgtgggtgttcgaagaagagatcaatggtgaaaaattgactgaaatcataaatactagacatcaaaacgtgaaatacttgcctggcatcactctacccgacaatttggttgctaatccagacttgattgattcagtcaaggatgtcgacatcatcgttttcaacattccacatcaatttttgccccgtatctgtagccaattgaaaggtcatgttgattcacacgtcagagctatctcctgtctaaagggttttgaagttggtgctaaaggtgtccaattgctatcctcttacatcactgaggaactaggtattcaatgtggtgctctatctggtgctaacattgccaccgaagtcgctcaagaacactggtctgaaacaacagttgcttaccacattccaaaggatttcagaggcgagggcaaggacgtcgaccataaggttctaaaggccttgttccacagaccttacttccacgttagtgtcatcgaagatgttgctggtatctccatctgtggtgctttgaagaacgttgttgccttaggttgtggtttcgtcgaaggtctaggctggggtaacaacgcttctgctgccatccaaagagtcggtttgggtgagatcatcagattcggtcaaatgtttttcccagaatctagagaagaaacatactaccaagagtctgctggtgttgctgatttgatcaccacctgcgctggtggtagaaacgtcaaggttgctaggctaatggctacttctggtaaggacgcctgggaatgtgaaaaggagttgttgaatggccaatccgctcaaggtttaattacctgcaaagaagttcacgaatggttggaaacatgtggctctgtcgaagacttcccattatttgaagccgtataccaaatcgtttacaacaactacccaatgaagaacctgccggacatgattgaagaattagatctacatgaagattagatttattggagaaagataacatatcatactttcccccacttttttcgaggctcttctatatcatattcataaattagcattatgtcatttctcataactactttatcacgttagaaattacttattattattaaattaatacaaaatttagtaaccaaataaatataaataaatatgtatatttaaattttaaaaaaaaaatcctatagagcaaaaggattttccattataatattagctgtacacctcttccgcattttttgagggtggttacaacaccactcattcagaggctgtcggcacagttgcttctagcatctggcgtccgtatgtatgggtgtattttaaataataaacaaagtgccacaccttcaccaattatgtctttaagaaatggacaagttccaaagagcttgcccaaggctcgacaaggatgtactttggaatatctatattcaagtacgtggcgcgcatatgtttgagtgtgcacacaataaaggtt

Sequence of gpd1Δ mutation (part of the gpd1 coding sequence was notdeleted (underlined) and the region that was deleted is represented by aΔ; SEQ ID NO:90):

tacaaacgcaacacgaaagaacaaaaaaagaagaaaacagaaggccaagacagggtcaatgagactgttgtcctcctactgtccctatgtctctggccgatcacgcgccattgtccctcagaaacaaatcaaacacccacaccccgggcacccaaagtccccacccacaccaccaatacgtaaacggggcgccccctgcaggccctcctgcgcgcggcctcccgccttgcttctctccccttccttttctttttccagttttccctattttgtccctttttccgcacaacaagtatcagaatgggttcatcaaatctatccaacctaattcgcacgtagactggcttggtattggcagtttcgtagttatatatatactaccatgagtgaaactgttacgttaccttaaattctttctccctttaattttcttttatcttactctcctacataagacatcaagaaacaattgtatattgtacaccccccccctccacaaacacaaatattgataatataaagatgtctgctgctgctgatagΔtctacatgaagattagatttattggagaaagataacatatcatactttcccccacttttttcgaggctcttctatatcatattcataaattagcattatgtcatttctcataactactttatcacgttagaaattacttattattattaaattaatacaaaatttagtaaccaaataaatataaataaatatgtatatttaaattttaaaaaaaaaatcctatagagcaaaaggattttccattataatattagctgtacacctcttccgcattttttgagggtggttacaacaccactcattcagaggctgtcggcacagttgcttctagcatctggcgtccgtatgtatgggtgtattttaaataataaacaaagtgccacaccttcaccaattatgtctttaagaaatggacaagttccaaagagcttgcccaaggctcgacaaggatgtactttggaatatctatattcaagtacgtggcgcgcatatgtttgagtgtgcacacaataaaggtt

Sequence of GPD2 locus (coding sequence is underlined; SEQ ID NO:91):

atagccatcatgcaagcgtgtatcttctaagattcagtcatcatcattaccgagtttgttttccttcacatgatgaagaaggtttgagtatgctcgaaacaataagacgacgatggctctgccattgttatattacgcttttgcggcgaggtgccgatgggttgctgaggggaagagtgtttagcttacggacctattgccattgttattccgattaatctattgttcagcagctcttctctaccctgtcattctagtattttttttttttttttttggttttacttttttttcttcttgcctttttttcttgttactttttttctagttttttttccttccactaagctttttccttgatttatccttgggttcttctttctactcctttagattttttttttatatattaatttttaagtttatgtattttggtagattcaattctctttccctttccttttccttcgctccccttccttatcaatgcttgctgtcagaagattaacaagatacacattccttaagcgaacgcatccggtgttatatactcgtcgtgcatataaaattttgccttcaagatctactttcctaagaagatcattattacaaacacaactgcactcaaagatgactgctcatactaatatcaaacagcacaaacactgtcatgaggaccatcctatcagaagatcggactctgccgtgtcaattgtacatttgaaacgtgcgcccttcaaggttacagtgattggttctggtaactgggggaccaccatcgccaaagtcattgcggaaaacacagaattgcattcccatatcttcgagccagaggtgagaatgtgggtttttgatgaaaagatcggcgacgaaaatctgacggatatcataaatacaagacaccagaacgttaaatatctacccaatattgacctgccccataatctagtggccgatcctgatcttttacactccatcaagggtgctgacatccttgttttcaacatccctcatcaatttttaccaaacatagtcaaacaattgcaaggccacgtggcccctcatgtaagggccatctcgtgtctaaaagggttcgagttgggctccaagggtgtgcaattgctatcctcctatgttactgatgagttaggaatccaatgtggcgcactatctggtgcaaacttggcaccggaagtggccaaggagcattggtccgaaaccaccgtggcttaccaactaccaaaggattatcaaggtgatggcaaggatgtagatcataagattttgaaattgctgttccacagaccttacttccacgtcaatgtcatcgatgatgttgctggtatatccattgccggtgccttgaagaacgtcgtggcacttgcatgtggtttcgtagaaggtatgggatggggtaacaatgcctccgcagccattcaaaggctgggtttaggtgaaattatcaagttcggtagaatgtttttcccagaatccaaagtcgagacctactatcaagaatccgctggtgttgcagatctgatcaccacctgctcaggcggtagaaacgtcaaggttgccacatacatggccaagaccggtaagtcagccttggaagcagaaaaggaattgcttaacggtcaatccgcccaagggataatcacatgcagagaagttcacgagtggctacaaacatgtgagttgacccaagaattcccattattcgaggcagtctaccagatagtctacaacaacgtccgcatggaagacctaccggagatgattgaagagctagacatcgatgacgaatagacactctccccccccctccccctctgatctttcctgttgcctctttttcccccaaccaatttatcattatacacaagttctacaactactactagtaacattactacagttattataattttctattctctttttctttaagaatctatcattaacgttaatttctatatatacataactaccattatacacgctattatcgtttacatatcacatcaccgttaatgaaagatacgacaccctgtacactaacacaattaaataatcgccataaccttttctgttatctatagcccttaaagctgtttcttcgagctttttcactgcagtaattctccacatgggcccagccactgagataagagcgctatgttagtcactactgacggctctccagtcatttatgtgattttttagtgactcatgtcgcatttggcccgtttttttccgctgtcgcaacctatttccattaacggtgccgtatggaagagtcatttaaaggcaggagagagagattactcatcttcattggatcagattgatgactgcgtacggcagat

Sequence of gpd2Δ mutation (the entire coding sequence was deleted,which is represented by a Δ; SEQ ID NO:92):

atagccatcatgcaagcgtgtatcttctaagattcagtcatcatcattaccgagtttgttttccttcacatgatgaagaaggtttgagtatgctcgaaacaataagacgacgatggctctgccattgttatattacgcttttgcggcgaggtgccgatgggttgctgaggggaagagtgtttagcttacggacctattgccattgttattccgattaatctattgttcagcagctcttctctaccctgtcattctagtattttttttttttttttttggttttacttttttttcttcttgcctttttttcttgttactttttttctagttttttttccttccactaagctttttccttgatttatccttgggttcttctttctactcctttagattttttttttatatattaatttttaagtttatgtattttggtagattcaattctctttccctttccttttccttcgctccccttccttatcΔctctgatctttcctgttgcctctttttcccccaaccaatttatcattatacacaagttctacaactactactagtaacattactacagttattataattttctattctctttttctttaagaatctatcattaacgttaatttctatatatacataactaccattatacacgctattatcgtttacatatcacatcaccgttaatgaaagatacgacaccctgtacactaacacaattaaataatcgccataaccttttctgttatctatagcccttaaagctgtttcttcgagctttttcactgcagtaattctccacatgggcccagccactgagataagagcgctatgttagtcactactgacggctctccagtcatttatgtgattttttagtgactcatgtcgcatttggcccgtttttttccgctgtcgcaacctatttccattaacggtgccgtatggaagagtcatttaaaggcaggagagagagattactcatcttcattgga tcagattgatgactgcgtacggcagat

Sequence of FDH1 locus (coding sequence is underlined; SEQ ID NO:93):

tatttttctatagatatttacactccgcaagtgcaaaaaaaaagcattatcgctaacgatcaagaggaactgagaccttattagttgtctttgttggcgtaacataaatttcttaggaaaagagaaaattatctcgaaggcaaaaataaaccaagcctcgagtttaatggttttctaaaaaacactttaaaaacagatcgccataaaaggagaagctccgtaggagaccgttttcgaaacctatgtagaaataaagggaaagctccaacggtttggataaatctttagaagcatagagtttatacaacattcagtacgaaatgtactctcgaaacgttctcttttcacggtgcttagtagcagaaaaaagtgtcggaaattacctattttgtcaccactcgaggataggcttgaaagagagttttaaccccaacttttctattttgcacttgtttggctatggtttaaaacattctgtttggaccaacagcccaagcggcttatcccttttctttttttcccttataatcgggaatttccttactaggaaggcaccgatactagaactccgaatgaaaaagacatgccagtaataaaactattttgatgttatgcggaatatactattcttggattattcactgttaactaaaagttggagaaatcactctgcactgtcaatcattgaaaaaaagaacatataaaagggcacaaaattgagtcttttttaatgagttcttgctgaggaaagtttagttaatatatcatttacgtaaaatatgcatattcttgtattgtgctttttttattcattttaagcaggaacaatttacaagtattgcaacgctaatcaaatcaaaataacagctgaaaattaatatgtcgaagggaaaggttttgctggttctttacgaaggtggtaagcatgctgaagagcaggaaaagttattggggtgtattgaaaatgaacttggtatcagaaatttcattgaagaacagggatacgagttggttactaccattgacaaggaccctgagccaacctcaacggtagacagggagttgaaagacgctgaaattgtcattactacgccctttttccccgcctacatctcgagaaacaggattgcagaagctcctaacctgaagctctgtgtaaccgctggcgtcggttcagaccatgtcgatttagaagctgcaaatgaacggaaaatcacggtcaccgaagttactggttctaacgtcgtttctgtcgcagagcacgttatggccacaattttggttttgataagaaactataatggtggtcatcaacaagcaattaatggtgagtgggatattgccggcgtggctaaaaatgagtatgatctggaagacaaaataatttcaacggtaggtgccggtagaattggatatagggttctggaaagattggtcgcatttaatccgaagaagttactgtactacgactaccaggaactacctgcggaagcaatcaatagattgaacgaggccagcaagcttttcaatggcagaggtgatattgttcagagagtagagaaattggaggatatggttgctcagtcagatgttgttaccatcaactgtccattgcacaaggactcaaggggtttattcaataaaaagcttatttcccacatgaaagatggtgcatacttggtgaataccgctagaggtgctatttgtgtcgcagaagatgttgccgaggcagtcaagtctggtaaattggctggctatggtggtgatgtctgggataagcaaccagcaccaaaagaccatccctggaggactatggacaataaggaccacgtgggaaacgcaatgactgttcatatcagtggcacatctctggatgctcaaaagaggtacgctcagggagtaaagaacatcctaaatagttacttttccaaaaagtttgattaccgtccacaggatattattgtgcagaatggttcttatgccaccagagcttatggacagaagaaataagagtgattatgagtatttgtgagcagaagttttccggtctccttttgttcttgttttggcgtattctccactattcgtccatagcacatttataccttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaaatagaagagcggtttattaatatcaaataattgaaactgctgatatggtagctatatacaaaatctgctgtcaaaatttggcagtaaacgatcttcacggtagcggttcaaataaagaggaaaagtctttctcccttactgtttttctggaatttggctcgtcgttaataacagaactaaagatacagtaaaaggagagatcgcaatcaacttcattaattgtaacagtagcataatcacaactgatcatctacactataaacagtttttatttctaattatgggcgcctggccggctcaaacattgtgcttttaagactccaaaagtatctgctgcagaaaagagccatataatgttaagtgttcagggataggttatcgcttactacttcaaacgtttcgaaggaaagccagggaagcctatatctgattccctgtttcataatccaatgcagccactagcttataattatttgaactatttgtcgaacatcacagtaataaaatccccagaaagttccacttgctgcatattggcacctgttgattcactctccatcacttttttgttagccgcccagcctagaaagtctttaaatacatctgaaatttttttttttttaacagtgcacccgtgcatcatacctcatgcaaggtacctttttttctcaaaggtattgtcttccattgaagtggcactatggcatgatgaaccctgagcatttctgaattcaacagaaccaaattgtccagaaataaatctgtccgacatgaattatgaaactttttttcaattaagtgaagagaattttgcagcgtcttaccattattttgacccattggtcgcatgtttgcgctttgacttcgagaaccatgttaaagcttacttgtacgacaaccaatgaagtatattacggcagtttttttggactgggtcaaaaaaagtgttgcataatcaaatcaggaacacattaaaatgttgtaaaatttgtcttagtatcacctgagtggttattcattacgtacta

Sequence of fdh1Δ mutation (the entire coding sequence was deleted,which is represented by a Δ; SEQ ID NO:94):

tatttttctatagatatttacactccgcaagtgcaaaaaaaaagcattatcgctaacgatcaagaggaactgagaccttattagttgtctttgttggcgtaacataaatttcttaggaaaagagaaaattatctcgaaggcaaaaataaaccaagcctcgagtttaatggttttctaaaaaacactttaaaaacagatcgccataaaaggagaagctccgtaggagaccgttttcgaaacctatgtagaaataaagggaaagctccaacggtttggataaatctttagaagcatagagtttatacaacattcagtacgaaatgtactctcgaaacgttctcttttcacggtgcttagtagcagaaaaaagtgtcggaaattacctattttgtcaccactcgaggataggcttgaaagagagttttaaccccaacttttctattttgcacttgtttggctatggtttaaaacattctgtttggaccaacagcccaagcggcttatcccttttctttttttcccttataatcgggaatttccttactaggaaggcaccgatactagaactccgaatgaaaaagacatgccagtaataaaactattttgatgttatgcggaatatactattcttggattattcactgttaactaaaagttggagaaatcactctgcactgtcaΔtggcagtaaacgatcttcacggtagcggttcaaataaagaggaaaagtctttctcccttactgtttttctggaatttggctcgtcgttaataacagaactaaagatacagtaaaaggagagatcgcaatcaacttcattaattgtaacagtagcataatcacaactgatcatctacactataaacagtttttatttctaattatgggcgcctggccggctcaaacattgtgcttttaagactccaaaagtatctgctgcagaaaagagccatataatgttaagtgttcagggataggttatcgcttactacttcaaacgtttcgaaggaaagccagggaagcctatatctgattccctgtttcataatccaatgcagccactagcttataattatttgaactatttgtcgaacatcacagtaataaaatccccagaaagttccacttgctgcatattggcacctgttgattcactctccatcacttttttgttagccgcccagcctagaaagtctttaaatacatctgaaatttttttttttttaacagtgcacccgtgcatcatacctcatgcaaggtacctttttttctcaaaggtattgtcttccattgaagtggcactatggcatgatgaaccctgagcatttctgaattcaacagaaccaaattgtccagaaataaatctgtccgacatgaattatgaaactttttttcaattaagtgaagagaattttgcagcgtcttaccattattttgacccattggtcgcatgtttgcgctttgacttcgagaaccatgttaaagcttacttgtacgacaaccaatgaagtatattacggcagtttttttggactgggtcaaaaaaagtgttgcataatcaaatcaggaacacattaaaatgttgtaaaatttgtcttagtatcacctgagtggttattcattacgtacta

Sequence of FDH2 locus (coding sequence is underlined; SEQ ID NO:95):

tgtcgagacaatgtcattgcaagttatataaacattgtaatacatcacctcgatgaaagagaaactggaatgatagatctctttttctcaaaatttcgttaatatgtaataataaggttcctgatgtaatttgtttttgtacaaattattttagattctggaggttcaaataaaatatatattacagccaacgattaggggggacaagacttgattacacatttttcgttggtaacttgactcttttatgaaaagaaaacattaagttgaaggtgcacgcttgaggcgctcctttttcatggtgcttagcagcagatgaaagtgtcagaagttacctattttgtcaccatttgagaataagcttgaaagaaagttgtaaccccaacttttctatcttgcacttgtttggaccaacagccaaacggcttatcccttttcttttcccttataatcgggaatttccttactaggaaggcaccgatactataactccgaatgaaaaagacatgccagtaataaaaataattgatgttatgcggaatatactattcttggattattcactgttaactaaaagttggagaaatcactctgcactgtcaatcattgaaaaaaagaacatataaaagggcacaaaatcgagtcttttttaatgagttcttgctgaggaaaatttagttaatatatcatttacataaaacatgcatattattgtgttgtactttctttattcattttaagcaggaataattacaagtattgcaacgctaatcaaatcgaaataacagctgaaaattaatatgtcgaagggaaaggttttgctggttctttatgaaggtggtaagcatgctgaagagcaggaaaagttattggggtgtattgaaaatgaacttggtatcagaaatttcattgaagaacagggatacgagttggttactaccattgacaaggaccctgagccaacctcaacggtagacagggagttgaaagacgctgaaattgtcattactacgccctttttccccgcctacatctcgagaaacaggattgcagaagctcctaacctgaagctctgtgtaaccgctggcgtcggttcagaccatgtcgatttagaagctgcaaatgaacggaaaatcacggtcaccgaagttactggttctaacgtcgtttctgtcgcagagcacgttatggccacaattttggttttgataagaaactataatggtggtcatcaataagcaattaatggtgagtgggatattgccggcgtggctaaaaaatgagtatgatctggaagacaaaataatttcaacggtaggtgccggtagaattggatatagggttctggaaagattggtcgcatttaatccgaagaagttactgtactacgactaccaggaactacctgcggaagcaatcaatagattgaacgaggccagcaagcttttcaatggcagaggtgatattgttcagagagtagagaaattggaggatatggttgctcagtcagatgttgttaccatcaactgtccattgcacaaggactcaaggggtttattcaataaaaagcttatttcccacatgaaagatggtgcatacttggtgaataccgctagaggtgctatttgtgtcgcagaagatgttgccgaggcagtcaagtctggtaaattggctggctatggtggtgatgtctgggataagcaaccagcaccaaaagaccatccctggaggactatggacaataaggaccacgtgggaaacgcaatgactgttcatatcagtggcacatctctgcatgctcaaaagaggtacgctcagggagtaaagaacatcctaaatagttacttttccaaaaagtttgattaccgtccacaggatattattgtgcagaatggttcttatgccaccagagcttatggacagaagaaataagagtgattatgagtatttgtgagcagaagttttccggtctccttttgttcttgttttggcgtattctccactattcgtccatagcacatttataccttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaaatagaagagcggtttattaatatcaaataattgaaactgctgatatggtagctatatacaaaatctgctgtcaaaatttggcagtaaacgatcttcacggtagcggttcaaataaagaggaaaagtccttctcccttactgtttttctggaatttggctcgtcgttaataacagaactaaagatacagtaaaaggagagatcgcaatcaacttcattaattgtaacagtagcataatcacaactggttatctgcgttatagacaattcttactcacaatgatgggcgcttagttggctgtaaacgtcgctttttaaaactccgaaaagttaccgctacagaaaaaaaccataaatgtatgctagttgcgcagagaggtttagggtccaaaatttactaccctgtcgctcactacagcgactgtcccgaattaagcccgaagagacgcagaactgttgtatgaacctcatgaaaccactgatcttgaagatttagaccttcagaatcgttttcaattagaagtatacaagaagtctttgtacaataatgtcaagacagagctctgaattatagttcagccttgttattttttttt

Sequence of fdh2Δ mutation (the entire coding sequence was deleted,which is represented by a Δ; SEQ ID NO:96):

tgtcgagacaatgtcattgcaagttatataaacattgtaatacatcacctcgatgaaagagaaactggaatgatagatctctttttctcaaaatttcgttaatatgtaataataaggttcctgatgtaatttgtttttgtacaaattattttagattctggaggttcaaataaaatatatattacagccaacgattaggggggacaagacttgattacacatttttcgttggtaacttgactcttttatgaaaagaaaacattaagttgaaggtgcacgcttgaggcgctcctttttcatggtgcttagcagcagatgaaagtgtcagaagttacctattttgtcaccatttgagaataagcttgaaagaaagttgtaaccccaacttttctatcttgcacttgtttggaccaacagccaaacggcttatcccttttcttttcccttataatcgggaatttccttactaggaaggcaccgatactataactccgaatgaaaaagacatgccagtaataaaaataattgatgttatgcggaatatactattcttggattattcactgttaactaaaagttggagaaatcactctgcactgtcaatcattgaaaaaaagaacatataaaagggcacaaaatcgagtcttttttaatgagttcttgctgaggaaaatttagttaatatatcatttacataaaacatgcatattattgtgttgtactttctttattcattttaagcaggaataattacaagtattgcaacgctaatcaaatcgaaataacagctgaaaattaatΔtaagagtgattatgagtatttgtgagcagaagttttccggtctccttttgttcttgttttggcgtattctccactattcgtccatagcacatttataccttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaaatagaagagcggtttattaatatcaaataattgaaactgctgatatggtagctatatacaaaatctgctgtcaaaatttggcagtaaacgatcttcacggtagcggttcaaataaagaggaaaagtccttctcccttactgtttttctggaatttggctcgtcgttaataacagaactaaagatacagtaaaaggagagatcgcaatcaacttcattaattgtaacagtagcataatcacaactggttatctgcgttatagacaattcttactcacaatgatgggcgcttagttggctgtaaacgtcgctttttaaaactccgaaaagttaccgctacagaaaaaaaccataaatgtatgctagttgcgcagagaggtttagggtccaaaatttactaccctgtcgctcactacagcgactgtcccgaattaagcccgaagagacgcagaactgttgtatgaacctcatgaaaccactgatcttgaagatttagaccttcagaatcgttttcaattagaagtatacaagaagtctttgtacaataatgtcaagacagagctctgaattatagttcagccttgttattttttttt

3.2 Generation of Glycerol-Reduced Strain Comprising gpd2Δ::GPD1

Glycerol-reduction strain gpd2Δ::GPD1, was constructed as describedabove. The sequence of gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ::GPD1/GPD1 is providedbelow.

Sequence of GPD1 at GPD2 locus (inserted GPD1 is underlined; SEQ IDNO:97):

agtaactgtgacgatatcaactctttttttattatgtaataagcaaacaagcacgaatggggaaagcctatgtgcaatcaccaaggtcgtcccttttttcccatttgctaatttagaatttaaagaaaccaaaagaatgaagaaagaaaacaaatactagccctaaccctgacttcgtttctatgataataccctgctttaatgaacggtatgccctagggtatatctcactctgtacgttacaaactccggttattttatcggaacatccgagcacccgcgccttcctcaacccaggcaccgcccccaggtaaccgtgcgcgatgagctaatcctgagccatcacccaccccacccgttgatgacagcaattcgggagggcgaaaaataaaaactggagcaaggaattaccatcaccgtcaccatcaccatcatatcgccttagcctctagccatagccatcatgcaagcgtgtatcttctaagattcagtcatcatcattaccgagtttgttttccttcacatgatgaagaaggtttgagtatgctcgaaacaataagacgacgatggctctgccattgttatattacgcttttgcggcgaggtgccgatgggttgctgaggggaagagtgtttagcttacggacctattgccattgttattccgattaatctattgttcagcagctcttctctaccctgtcattctagtattttttttttttttttttggttttacttttttttcttcttgcctttttttcttgttactttttttctagttttttttccttccactaagctttttccttgatttatccttgggttcttctttctactcctttagattttttttttatatattaatttttaagtttatgtattttggtagattcaattctctttccctttccttttccttcgctccccttccttatcaatgtctgctgctgctgatagattaaacttaacttccggccacttgaatgctggtagaaagagaagttcctcttctgtttctttgaaggctgccgaaaagcctttcaaggttactgtgattggatctggtaactggggtactactattgccaaggtggttgccgaaaattgtaagggatacccagaagttttcgctccaatagtacaaatgtgggtgttcgaagaagagatcaatggtgaaaaattgactgaaatcataaatactagacatcaaaacgtgaaatacttgcctggcatcactctacccgacaatttggttgctaatccagacttgattgattcagtcaaggatgtcgacatcatcgttttcaacattccacatcaatttttgccccgtatctgtagccaattgaaaggtcatgttgattcacacgtcagagctatctcctgtctaaagggttttgaagttggtgctaaaggtgtccaattgctatcctcttacatcactgaggaactaggtattcaatgtggtgctctatctggtgctaacattgccaccgaagtcgctcaagaacactggtctgaaacaacagttgcttaccacattccaaaggatttcagaggcgagggcaaggacgtcgaccataaggttctaaaggccttgttccacagaccttacttccacgttagtgtcatcgaagatgttgctggtatctccatctgtggtgctttgaagaacgttgttgccttaggttgtggtttcgtcgaaggtctaggctggggtaacaacgcttctgctgccatccaaagagtcggtttgggtgagatcatcagattcggtcaaatgtttttcccagaatctagagaagaaacatactaccaagagtctgctggtgttgctgatttgatcaccacctgcgctggtggtagaaacgtcaaggttgctaggctaatggctacttctggtaaggacgcctgggaatgtgaaaaggagttgttgaatggccaatccgctcaaggtttaattacctgcaaagaagttcacgaatggttggaaacatgtggctctgtcgaagacttcccattatttgaagccgtataccaaatcgtttacaacaactacccaatgaagaacctgccggacatgattgaagaattagatctacatgaagattagacactctccccccccctccccctctgatctttcctgttgcctctttttcccccaaccaatttatcattatacacaagttctacaactactactagtaacattactacagttattataattttctattctctttttctttaagaatctatcattaacgttaatttctatatatacataactaccattatacacgctattatcgtttacatatcacatcaccgttaatgaaagatacgacaccctgtacactaacacaattaaataatcgccataaccttttctgttatctatagcccttaaagctgtttcttcgagctttttcactgcagtaattctccacatgggcccagccactgagataagagcgctatgttagtcactactgacggctctccagtcatttatgtgattttttagtgactcatgtcgcatttggcccgtttttttccgctgtcgcaacctatttccattaacggtgccgtatggaagagtcatttaaaggcaggagagagagattactcatcttcattggatcagattgatgactgcgtacggcagatagtgtaatctgagcagttgcgagacccagactggcactgtctcaatagtatattaatgggcatacattcgtactcccttgttcttgcccacagttctctctctctttacttcttgtatcttgtctccccattgtgcagcgataaggaacattgttctaatatacacggatacaaaagaaatacacat

Example 4 Cloning and Characterization of PFL and AADH Enzymes

To identify PFL enzymes for use in the strains of the invention, severalPFL enzymes were identified for cloning and functional analysis. SeeTable 1. Functionality was determined by plasmid based expression ofeach PFL in the fcyΔ::ADHE gpd1Δ::ADHE gpd2Δfdh1Δfdh2Δ (M2158)background. FIG. 22 shows fermentation performance in 20% corn mash. APFL was determined to be functional based on the presence or absence ofa yield increase over M2085. The C. cellulolyticum PFL was determined tobe non-functional based on data shown in FIG. 13. The strain listed asM1992+pMU2481 is M2085 plus a plasmid expressing the C. cellulolyticumPFL. This strain does not appear to make formate.

TABLE 1 Analysis of PFL Enzymes Organism Functional SEQ ID NOs: Bacilluslicheniformis ATCC_14580 nd  6 and 40 Streptococcus thermophilus LMD_9nd 12 and 46 Lactobacillus plantarum WCFS1 nd 16 and 50 (lp_3314 andlp_3313) Lactobacillus casei ATCC_334 yes 24 and 58 Bifidobacteriumadolescentis yes 26 and 60 Clostridium cellulolyticum no 34 and 68Escherichia coli yes 36 and 70 Chlamydomonas reinhardtii PflA yes 72 and76 Piromyces sp. E2 yes 78 Neocallimastix frontalis yes 74 and 80

As shown in Example 7, eight of nine PFL enzymes that were tested canenable the glycerol elimination and glycerol reduction technologiesdescribed herein. An alignment of six of these PFL enzymes is shown inFIG. 12. Four of the residues are absent in C. cellulolyticum yetconserved among the other PFL enzymes (indicated with asterisks in FIG.12). There is an insertion of 18 amino acids at position 640 of the B.adolescentis PFL, which is not present in the other PFL enzymes. Theeukaryotic PFLs (Piromyces and Chlamydomonas) have an N-terminalextension which has been reported to be involved in mitochondrialtargeting. Deletion of this sequence may improve the performance ofthese enzymes in S. cerevisiae. These differences may provide insightsinto identifying additional PFL enzymes for use in the strains of theinvention.

To identify AADH enzymes for use in the strains of the invention,several AADH enzymes were identified for cloning and functionalanalysis. See Table 2. Functionality was determined though analysis ofthe data listed in Table 3 below and shown in FIG. 20. An AADH enzymewas determined to be functional if a strain containing the genotypegpd1Δgpd2Δ plus a given AADH, had a faster anaerobic growth rate thanstrain gpd1Δgpd2Δ (FIG. 20) and there was evidence for acetateconsumption (Table 3).

TABLE 2 Analysis of AADH Enzymes Organism Functional SEQ ID NO:Escherichia coli yes 84 Clostridium phytofermentans yes 82 Chlamydomonasreinhardtii yes 86 Piromyces sp. E2 yes 88 Bifidobacterium adolescentisyes 100

When glycerol deletion strains are grown anaerobically, they are notcapable of growth or fermentation and cannot consume sugar duringglycolysis. However, if these glycerol deletion strains are complementedwith an AADH, the strains are able to grow with the supplementation ofacetate in the media. FIG. 20 shows the growth rates of the parentalstrain, the glycerol deletion strain, and four glycerol deletion strainsexpressing AADHs from Escherichia coli (Eco), Clostridiumphytofermentans (Cph), Chlamydomonas reinhardtii (Chl), and Piromycessp. E2 (Pir). As shown in FIG. 20, all four genes can restore growthlevels above the glycerol deletion strain (as noted by the dotted line)indicating a functional AADH.

The product yields and conversion of acetate by the strains above, aswell as additional strains, are shown in Table 3. The glycerol deletionstrain was unable to consume sugar or produce ethanol. The parent strainproduced glycerol and ethanol but was unable to convert the acetate inthe media, initially present at ˜2 g/L, giving an ethanol yield of 0.41g/g glucose, consistent with anaerobic ethanol yields. The glyceroldeletion strains complemented with AADHs, however, were able to consumeglucose and produce ethanol without producing glycerol, or the glycerolproduction was significantly decreased compared to the parent strain(Chl AADH). See Table 3. In these glycerol deletion mutants, the acetatelevels were also reduced, resulting in higher ethanol yields (calculatedas grams ethanol produced per gram consumed glucose) than was achievedby the parent strain.

TABLE 3 Product Yields and Acetate Conversion of Glycerol DeletionStrains Expressing AADH Acetate Ethanol Growth Strain Glycerol^(a)Uptake^(a) Ethanol^(a) Yield^(b) rate (hr⁻¹) Parent M139 1.37 0.14 10.410.42 0.27 Δgpd1Δgpd2 0.00 0.00 0.00 0.00 0.01 M2032 Eco AADH 0.00 0.6211.39 0.46 0.17 M1991 Cph AADH 0.00 0.66 11.21 0.45 0.18 M1991 Chl AADH0.00 0.32 9.04 0.47 0.04 M1991 Pir AADH 0.00 0.68 11.17 0.45 0.17 M1991Bad AADH 0.00 0.67 10.95 0.44 0.18 M1991 Eco mhpF 0.00 0.03 0.50 0.020.06 M1991 Cph ADH 0.00 0.60 11.29 0.45 0.19 (1428) M1991 Cph ADH 0.000.74 11.22 0.45 0.20 (2642) M1991 Tsac AADH 0.00 0.59 11.89 0.48 0.16M1991 ^(a)grams per liter ^(b)gram ethanol produced per gram sugarconsumed

Example 5 Expression of PFL and AADH and Detection of Formate

To examine the expression of formate in a yeast strain of the invention,E. coli PFL was cloned and expressed in an FDH deletion strain. StrainM1992+pMU2483 has deletions of FDH1 and FDH2 and a plasmid expressingthe E. coli PflA and PflB. This strain was constructed by transformingstrain M1992 (fdh1Δfdh2Δ) with plasmids expressing either C.cellulolyticum PFL (pMU2481) or E. coli PFL (pMU2483).

The strains were grown in YNB medium buffered with HEPES at pH 6.5, andformate was measured using a formate detection kit from Megazymes (Cat.No. K-FORM), according to manufacturer's specifications. As shown inFIG. 13, approximately 0.0125 g/L and 0.023 g/L formate was measuredafter 24 hours and 48 hours of growth, respectively. Similar resultshave been achieved by overexpressing an E. coli PFL in S. cerevesiae.Waks and Silver, Appl. Env. Microbiol. 75:1867-75 (2009).

PFL was also co-expressed in a strain expressing AADH. See FIG. 14.M2085 is a background strain with the genotype gpd1Δgpd2Δfdh1Δfdh2Δ andM2032 has the genotype gpd1Δgpd2Δ. Both of these strains are unable togrow anaerobically even in the presence of acetate. M2158 was created byintegrating multiple copies of E. coli AADHs at the GPD1 and FCY1 locusin the M2085 background. The integration schemes are shown in FIGS. 26and 27 and the corresponding nucleotide sequences for M2158 are listedbelow. One copy of AADH is driven by the native GPD1 promoter. See FIG.26. The second copy is oriented in the reverse direction and is drivenby the phosphofructokinase promoter. See FIG. 26. For AADH integrationin FCY1, one copy is driven by the ENOL promoter and a second copy isdriven by the PFK1 promoter. See FIG. 27. M2182 was created bytransforming a vector expressing the B. adolescentis PFLs into the M2158background.

Sequence of M2158 AADH integrations at the GPD1 locus (nucleotide; SEQID NO:98):

tagattcttttcgaatttgtggtgaagataggaaagttggtacagttctccatcaattttccatattttgctaaaaactcccttgcatgtctctttgcattcatttctcctgtatacgggttcaacacatcaatcgaattttgcaaagttgtctccatttctagaagactttcatcgggaataaaaaattcatatccattattcaaaaacgataatgatccctcgtacttacctgtgtaattggatattttataccatacttcaaaaatatccttggcctcacttctggtaggatacctttcgccatgtctgccaatcatttgaacttgcgttaatctacaaccttcaggaatatcagtgggtataccgtagttagcgggaaaggagaaatatggcgcagaccctccaagaaagggaaacagactcttctgagagccaattagttcaatatccgcaaaacttctgagtgggatggagagtgccttagataatagaacacctaaacaaatggcaaaaataacgggcttcaccattgttcctgtatggtgtattagaacatagctgaaaatacttctgcctcaaaaaagtgttaaaaaaaagaggcattatatagaggtaaagcctacaggcgcaagataacacatcaccgctctcccccctctcatgaaaagtcatcgctaaagaggaacactgaaggttcccgtaggttgtctttggcacaaggtagtacatggtaaaaactcaggatggaataattcaaattcaccaatttcaacgtcccttgtttaaaaagaaaagaatttttctctttaaggtagcactaatgcattatcgatgatgtaaccattcacacaggttatttagcttttgatccttgaaccattaattaacccagaaatagaaattacccaagtggggctctccaacacaatgagaggaaaggtgactttttaagggggccagaccctgttaaaaacctttgatggctatgtaataatagtaaattaagtgcaaacatgtaagaaagattctcggtaacgaccatacaaatattgggcgtgtggcgtagtcggtagcgcgctcccttagcatgggagaggtctccggttcgattccggactcgtccaaattattttttactttccgcggtgccgagatgcagacgtggccaactgtgtctgccgtcgcaaaatgatttgaattttgcgtcgcgcacgtttctcacgtacataataagtattttcatacagttctagcaagacgaggtggtcaaaatagaagcgtcctatgttttacagtacaagacagtccatactgaaatgacaacgtacttgacttttcagtattttctttttctcacagtctggttatttttgaaagcgcacgaaatatatgtaggcaagcattttctgagtctgctgacctctaaaattaatgctattgtgcaccttagtaacccaaggcaggacagttaccttgcgtggtgttactatggccggaagcccgaaagagttatcgttactccgattattttgtacagctgatgggaccttgccgtcttcattttttttttttttcacctatagagccgggcagagctgcccggcttaactaagggccggaaaaaaaacggaaaaaagaaagccaagcgtgtagacgtagtataacagtatatctgacacgcacgtgatgaccacgtaatcgcatcgcccctcacctctcacctctcaccgctgactcagcttcactaaaaaggaaaatatatactctttcccaggcaaggtgacagcggtccccgtctcctccacaaaggcctctcctggggtttgagcaagtctaagtttacgtagcataaaaattctcggattgcgtcaaataataaaaaaagtaaccccacttctacttctacatcggaaaaacattccattcacatatcgtctttggcctatcttgttttgtcctcggtagatcaggtcagtacaaacgcaacacgaaagaacaaaaaaagaagaaaacagaaggccaagacagggtcaatgagactgttgtcctcctactgtccctatgtctctggccgatcacgcgccattgtccctcagaaacaaatcaaacacccacaccccgggcacccaaagtccccacccacaccaccaatacgtaaacggggcgccccctgcaggccctcctgcgcgcggcctcccgccttgcttctctccccttccttttctttttccagttttccctattttgtccctttttccgcacaacaagtatcagaatgggttcatcaaatctatccaacctaattcgcacgtagactggcttggtattggcagtttcgtagttatatatatactaccatgagtgaaactgttacgttaccttaaattctttctccctttaattttcttttatcttactctcctacataagacatcaagaaacaattgtatattgtacaccccccccctccacaaacacaaatattgataatataaagatggctgttactaatgtcgctgaacttaacgcactcgtagagcgtgtaaaaaaagcccagcgtgaatatgccagtttcactcaagagcaagtagacaaaatcttccgcgccgccgctctggctgctgcagatgctcgaatcccactcgcgaaaatggccgttgccgaatccggcatgggtatcgtcgaagataaagtgatcaaaaaccactttgcttctgaatatatctacaacgcctataaagatgaaaaaacctgtggtgttctgtctgaagacgacacttttggtaccatcactatcgctgaaccaatcggtattatttgcggtatcgttccgaccactaacccgacttcaactgctatcttcaaatcgctgatcagtctgaagacccgtaacgccattatcttctccccgcacccgcgtgcaaaagatgccaccaacaaagcggctgatatcgttctgcaggctgctatcgctgccggtgctccgaaagatctgatcggctggatcgatcaaccttctgttgaactgtctaacgcactgatgcaccacccagacatcaacctgatcctcgcgactggtggtccgggcatggttaaagccgcatacagctccggtaaaccagctatcggtgtaggcgcgggcaacactccagttgttatcgatgaaactgctgatatcaaacgtgcagttgcatctgtactgatgtccaaaaccttcgacaacggcgtaatctgtgcttctgaacagtctgttgttgttgttgactctgtttatgacgctgtacgtgaacgttttgcaacccacggcggctatctgttgcagggtaaagagctgaaagctgttcaggatgttatcctgaaaaacggtgcgctgaacgcggctatcgttggtcagccagcctataaaattgctgaactggcaggcttctctgtaccagaaaacaccaagattctgatcggtgaagtgaccgttgttgatgaaagcgaaccgttcgcacatgaaaaactgtccccgactctggcaatgtaccgcgctaaagatttcgaagacgcggtagaaaaagcagagaaactggttgctatgggcggtatcggtcatacctcttgcctgtacactgaccaggataaccaaccggctcgcgtttcttacttcggtcagaaaatgaaaacggcgcgtatcctgattaacaccccagcgtctcagggtggtatcggtgacctgtataacttcaaactcgcaccttccctgactctgggttgtggttcttggggtggtaactccatctctgaaaacgttggtccgaaacacctgatcaacaagaaaaccgttgctaagcgagctgaaaacatgttgtggcacaaacttccgaaatctatctacttccgccgtggctccctgccaatcgcgctggatgaagtgattactgatggccacaaacgtgcgctcatcgtgactgaccgcttcctgttcaacaatggttatgctgatcagatcacttccgtactgaaagcagcaggcgttgaaactgaagtcttcttcgaagtagaagcggacccgaccctgagcatcgttcgtaaaggtgcagaactggcaaactccttcaaaccagacgtgattatcgcgctgggtggtggttccccgatggacgccgcgaagatcatgtgggttatgtacgaacatccggaaactcacttcgaagagctggcgctgcgctttatggatatccgtaaacgtatctacaagttcccgaaaatgggcgtgaaagcgaaaatgatcgctgtcaccaccacttctggtacaggttctgaagtcactccgtttgcggttgtaactgacgacgctactggtcagaaatatccgctggcagactatgcgctgactccggatatggcgattgtcgacgccaacctggttatggacatgccgaagtccctgtgtgctttcggtggtctggacgcagtaactcacgccatggaagcttatgtttctgtactggcatctgagttctctgatggtcaggctctgcaggcactgaaactgctgaaagaatatctgccagcgtcctaccacgaagggtctaaaaatccggtagcgcgtgaacgtgttcacagtgcagcgactatcgcgggtatcgcgtttgcgaacgccttcctgggtgtatgtcactcaatggcgcacaaactgggttcccagttccatattccgcacggtctggcaaacgccctgctgatttgtaacgttattcgctacaatgcgaacgacaacccgaccaagcagactgcattcagccagtatgaccgtccgcaggctcgccgtcgttatgctgaaattgccgaccacttgggtctgagcgcaccgggcgaccgtactgctgctaagatcgagaaactgctggcatggctggaaacgctgaaagctgaactgggtattccgaaatctatccgtgaagctggcgttcaggaagcagacttcctggcgaacgtggataaactgtctgaagatgcattcgatgaccagtgcaccggcgctaacccgcgttacccgctgatctccgagctgaaacagattctgctggatacctactacggtcgtgattatgtagaaggtgaaactgcagcgaagaaagaagctgctccggctaaagctgagaaaaaagcgaaaaaatccgcttaagtcgagagcttttgattaagccttctagtccaaaaaacacgtttttttgtcatttatttcattttcttagaatagtttagtttattcattttatagtcacgaatgttttatgattctatatagggttgcaaacaagcatttttcattttatgttaaaacaatttcaggtttaccttttattctgcttgtggtgacgcgtgtatccgcccgctcttttggtcacccatgtatttaattgcataaataattcttaaaagtggagctagtctatttctatttacatacctctcatttctcatttcctcctaatgtgtcaatgatcatattcttaactggaccgatcttattcgtcagattcaaaccaaaagttcttagggctaccacaggaggaaaattagtgtgatataatttaaataatttatccgccattcctaatagaacgttgttcgacggatatctttctgcccaaaagggttctaagctcaatgaagagccaatgtctaaacctcgttacattgaaaatacagtaaatggttccaccattattatgttggtcttgtttagtatggccgatcggcgtgtgttttgtttgcaccttttatatagtagaagaatatttgtcttaattcttattagtactgcaacctaaccactaattatcaacaattattggattatataaaggaggtaaattgccggattaaaatcaaatatcattcatcaacaagtattcatattgtcggcatatttttacatgcggtgtaagtatttggatcgtattcttatagtgtcaatacctcgaagcagcgtttcaagtaccagacgtatgtaggaactttttaacgtcgagtccgtaagatttgatcagtattaaaaaaatctagataaatgagtggtacaaataaaaacatcattaaaaatcgttaaataaaaaagtatgaagatcatctattaaagtattagtagccattagccttaaaaaaatcagtgctagtttaagtataatctcgggcgcgccggccgaggcggttaagcggattttttcgcttttttctcagctttagccggagcagcttctttcttcgctgcagtttcaccttctacataatcacgaccgtagtaggtatccagcagaatctgtttcagctcggagatcagcgggtaacgcgggttagcgccggtgcactggtcatcgaatgcatcttcagacagtttatccacgttcgccaggaagtctgcttcctgaacgccagcttcacggatagatttcggaatacccagttcagctttcagcgtttccagccatgccagcagtttctcgatcttagcagcagtacggtcgcccggtgcgctcagacccaagtggtcggcaatttcagcataacgacggcgagcctgcggacggtcatactggctgaatgcagtctgcttggtcgggttgtcgttcgcattgtagcgaataacgttacaaatcagcagggcgtttgccagaccgtgcggaatatggaactgggaacccagtttgtgcgccattgagtgacatacacccaggaaggcgttcgcaaacgcgatacccgcgatagtcgctgcactgtgaacacgttcacgcgctaccggatttttagacccttcgtggtaggacgctggcagatattctttcagcagtttcagtgcctgcagagcctgaccatcagagaactcagatgccagtacagaaacataagcttccatggcgtgagttactgcgtccagaccaccgaaagcacacagggacttcggcatgtccataaccaggttggcgtcgacaatcgccatatccggagtcagcgcatagtctgccagcggatatttctgaccagtagcgtcgtcagttacaaccgcaaacggagtgacttcagaacctgtaccagaagtggtggtgacagcgatcattttcgctttcacgcccattttcgggaacttgtagatacgtttacggatatccataaagcgcagcgccagctcttcgaagtgagtttccggatgttcgtacataacccacatgatcttcgcggcgtccatcggggaaccaccacccagcgcgataatcacgtctggtttgaaggagtttgccagttctgcacctttacgaacgatgctcagggtcgggtccgcttctacttcgaagaagacttcagtttcaacgcctgctgctttcagtacggaagtgatctgatcagcataaccattgttgaacaggaagcggtcagtcacgatgagcgcacgtttgtggccatcagtaatcacttcatccagcgcgattggcagggagccacggcggaagtagatagatttcggaagtttgtgccacaacatgttttcagctcgcttagcaacggttttcttgttgatcaggtgtttcggaccaacgttttcagagatggagttaccaccccaagaaccacaacccagagtcagggaaggtgcgagtttgaagttatacaggtcaccgataccaccctgagacgctggggtgttaatcaggatacgcgccgttttcattttctgaccgaagtaagaaacgcgagccggttggttatcctggtcagtgtacaggcaagaggtatgaccgataccgcccatagcaaccagtttctctgctttttctaccgcgtcttcgaaatctttagcgcggtacattgccagagtcggggacagtttttcatgtgcgaacggttcgctttcatcaacaacggtcacttcaccgatcagaatcttggtgttttctggtacagagaagcctgccagttcagcaattttataggctggctgaccaacgatagccgcgttcagcgcaccgtttttcaggataacatcctgaacagctttcagctctttaccctgcaacagatagccgccgtgggttgcaaaacgttcacgtacagcgtcataaacagagtcaacaacaacaacagactgttcagaagcacagattacgccgttgtcgaaggttttggacatcagtacagatgcaactgcacgtttgatatcagcagtttcatcgataacaactggagtgttgcccgcgcctacaccgatagctggtttaccggagctgtatgcggctttaaccatgcccggaccaccagtcgcgaggatcaggttgatgtctgggtggtgcatcagtgcgttagacagttcaacagaaggttgatcgatccagccgatcagatctttcggagcaccggcagcgatagcagcctgcagaacgatatcagccgctttgttggtggcatcttttgcacgcgggtgcggggagaagataatggcgttacgggtcttcagactgatcagcgatttgaagatagcagttgaagtcgggttagtggtcggaacgataccgcaaataataccgattggttcagcgatagtgatggtaccaaaagtgtcgtcttcagacagaacaccacaggttttttcatctttataggcgttgtagatatattcagaagcaaagtggtttttgatcactttatcttcgacgatacccatgccggattcggcaacggccattttcgcgagtgggattcgagcatctgcagcagccagagcggcggcgcggaagattttgtctacttgctcttgagtgaaactggcatattcacgctgggctttttttacacgctctacgagtgcgttaagttcagcgacattagtaacagccataattcttaattaactttgatatgattttgtttcagattttttatataaaagctttcccaaatagtgctaaagtgaacttagattttttggtacctgtttcgaaattaaaaatagaaaaatttctctccctatattgttattcttacttcaaatttgtttatcgtttatttactaggcgagacttgagtagacgacaatccaaatagaattaacagattttattggtagaaagcaataatattctttagatggttgagaataaagaagtaaaaaaaccagtaaagagaaaaagaaaaggaagaaaattaaagaaaaaggatgattacacaagaagataataaaaaaactcctttattaagagcggaagaatttaataatgaagatgggaataagcaaaacaaaaacaaagaagggaaaaaaaataaaaaatcgtatttatttatttaaaaaatcatgttgatgacgacaatggaaaaaaaaaaccgatttcactttctcatccttatatttttcaaaggttgatgcaagtcgatctcaaatcggataacgctgccaactgggaaattccgcaattccgcaagaaaaaaaaaaatgtgaaaacgtgattgcattttttacaggtcctaaaggatttagcccacatatcaagagggtggcagtaattgcactgattaagcattcgtcagcattaggcgaatgtgtgcatgaatattgccagtgtgctcgatattagagagtacattgaagaatattgtaccggattatgtacaataactttgttaatgagatattaattttcttttttactagccgctatcccatgcacgatgctaaatttcaagaagaaactgagatttaaaaaattagtggaagctgataaaacggactataatggtgtatggattgaggaatctcgacatgtttttccatcgttttcaacgatgactgtaacccgtagattgaaccaggcatgccaaagttagttagatcagggtaaaaattatagatgaggtatttattggagaaagataacatatcatactttcccccacttttttcgaggctcttctatatcatattcataaattagcattatgtcatttctcataactactttatcacgttagaaattacttattattattaaattaatacaaaatttagtaaccaaataaatataaataaatatgtatatttaaattttaaaaaaaaaatcctatagagcaaaaggattttccattataatattagctgtacacctcttccgcattttttgagggtggttacaacaccactcattcagaggctgtcggcacagttgcttctagcatctggcgtccgtatgtatgggtgtattttaaataataaacaaagtgccacaccttcaccaattatgtctttaagaaatggacaagttccaaagagcttgcccaaggctcgacaaggatgtactttggaatatctatattcaagtacgtggcgcgcatatgtttgagtgtgcacacaataaaggtttttagatattttgcggcgtcctaagaaaataaggggtttcttaaaaaataacaatagcaaacaaagttccttacgatgatttcagatgtgaatagcatggtcatgatgagtatatacgtttttataaataattaaaagttttcctcttgtctgtttttttgttggctcgtggttgttctcgaaaaaggagagttttcattttcgaaataggtgattatcatcatgttgttatcaccccacgacgaagataatacggagctcaccgttttctttttttttccctttggctgaaatttcccaccagaacaaacgtgacaaaattatctttgaatccaaagtagcttatatatatacgtagaagtgtttcgagacacacatccaaatacgaggttgttcaatttaaacccaagaatacataaaaaaaatatagatatattaacttagtaaacaatgactgcaagcacaccatccaatgtcatgacattgttcttgttaaggcatggacaaagtgaattgaatcacgagaatatattctgtggttggattgacgctaagctaaccgaaaaaggtaaagaacaagctcgtcattctgccgagctaatcgaacaatattgtaaagctaataatttgagattaccccagattggttacacctcacgtttaattaggacccaacagaccatagaaacgatgtgtgaagaatttaagttaaagccacaactgcaggttgtttacgactttaataaaatcaaacttggagacgaatttggcagtgatgacaaggataatatgaaaatcccgattcttcaaacttggaggctaaatgaacgtcattacggttcctggcagggccagaggaaaccgaatgttttaaaagaatatggtaaggataaatatatgttcattaggagagattacgagggtaagccaccacctgtagatcttgaccgtgagatgattcaacaagaaaatgagaagggctcttctactgggtacgaattcaaggagccaaacagacaaataaaatatgaattggaatgcagcaatcatgacattgtattaccggattccgaatctcttcgtgaagtggtttatagattgaatccttttctacaaaatgtcatattaaaattagccaatcaatatgatgaatcttcatgcctgattgtgggccatggaagttcagtgagatcgctactgaaaattctggagggtatatcagatgatgacatcaagaatgttgatattccaaatggtatccccttagtcgttgaattagataagaataatggtcttaagtttatcagaaaattctacctagatcctgaatctgctaagatcaatgctgagaaagtccgtaatgagggtttcataaaaaatccttaaggggggtaagtatataatataattgtgtattttccgaagtataatgaaaaccaatagaaaacttattataagtccaatgaggtactttaaaaatgtgatatttataagaacattcctgaatgcagatatatgatatatattgtaaatatatatagatgtgtatatgtatttccattttgtgtgaggttttcttcttttatctcctatataatttgtaaccttaattaacccatgacataaccaatattagcctttgcaaattttgtaacttcttgacgttgttctaacgacaaatcttcatgcttcgattttatatgccttgttaaagcatccagtctcgaaaacgtcttctgatagccctcagatccaagaatttttatacactccgagcaacggaagacaatcttccttttagcgtgaatggtattttggtgtctcgttaaatcataggaccttgaaaattgggcaccacacggttcatttgtaatgagattcattatctgacacgtaaatatttcgttattaccgtcagaagatgacgtatgggccgatggtgatgcagtcgaaggtttcgaattcgaatttgtagatgaatgtgaagataagtgcttc

Sequence of M2158 AADH integrations at the GPD1 locus (nucleotide; SEQID NO:99):

cagaattggtgatattcttcattatcccctcgacatctttacttttgatcagctttgtgtatagcgggatatccgattggaacttggcttcagcaacaaacttgccaaagtggattctcctactcaagctttgcaaacattctatatctctagtggcaacagaaccgaagttattcttatcatcaccatctcttttcgaaattaatggtataatcttttcaatataaactttttttattttatcattgtaattaacttctggggcataaggcgccaaaatttgtgggtagttaatgctcggtaagaatgatttctgaatcttgtcaggaaagaagggagtttcatcaggtgattcgaatcttctgatgcgagaatgcgcaatttcaagatttgaaagagcccaatccaagaaagatcctttaaaattcggaatttctaaacctggatggtttgcctcataaactgaaggacatgtggcgaaatgcgacctctcaataaatttgaagatgatcgaatcctccattctaactaattcatctctaatattttgtagatttaaaacagtttctggttttgtgaaatccatatcttataccaattttatgcaggatgctgagtgctatttgttagcaaaacggaatcgatgtttcaacttttcaatactttttttcttcttttcttcgaacttcgaagattatagagatccattgaaaatttacgataaaggaaatgcaacacgaagtttgaaaaaaagttgatattgaaaaaaaaaaaaaaagagaaccaaaaaaaaattaaaaaacgtgaaaacgatggtttaacaacttttttcgaatttggtatacgtggaaaaacgaatgtatagatgcatttttaaagaatatatataaaatttagtaattgtattccgcgagcggcgcaataggtgatttcatttagctgctttcagtttgcgtatcatttcttcattgatcttggcttctctatctaaatcctctttcgacttgtaaagtccccaagttctaaaccataagaaccgcctcaatctggaaaatttgtcagtatcaagaccataattcgtgtatgactgaatcaaatgtaatccactttcgtcatgagtaaattcggccttgctcagagactcctggattttggctaacaacgcagtcccttcgatgcatatagctaggccacaaattatgccaataacggtccatgggttgatgttttcttgaattctttcgtttttcatgctatttgcgtcttcccaagtcccagcgttccagtattcatactgcgcgttagagtggtagccataagagccggcatattggtaattttcagtattaacgttagaacgtggtgaatacgatgtggtccagccttgcctcgttgtgtcatatacgatctttttctttgggtcacaaagaatatcatatgcttgagagatgactttaaatctatgtagtttttcgcttgatgttagcagcagcggtgatttactatcactgttggtaaccttttctgagctaaatatttgaatgttatcggaatggtcagggtggtacaattttacataacgatgatattttttttttaacgacttcttgtccagtttaggatttccagatccggcctttggaatgccaaaaatatcatagggagttggatctgccaactcaggccattgttcatcccttatcgtaagttttctattgccatttttatcgttcgctgtagcatacttagctataaaagtgatttgtgggggacacttttctacacatgataagtgccacttgaataaaaatgggtatacgaacttatggtgtagcataacaaatatattgcaagtagtgacctatggtgtgtagatatacgtacagttagttacgagcctaaagacacaacgtgtttgttaattatactgtcgctgtaatatcttctcttccattatcaccggtcattccttgcaggggcggtagtacccggagaccctgaacttttctttttttttttgcgaaattaaaaagttcattttcaattcgacaatgagatctacaagccattgttttatgttgatgagagccagcttaaagagttaaaaatttcatagctactagtcttctaggcgggttatctactgatccgagcttccactaggatagcacccaaacacctgcatatttggacgacctttacttacaccaccaaaaaccactttcgcctctcccgcccctgataacgtccactaattgagcgattacctgagcggtcctcttttgtttgcagcatgagacttgcatactgcaaatcgtaagtagcaacgtctcaaggtcaaaactgtatggaaaccttgtcacctcacttaattctagctagcctaccctgcaagtcaagaggtctccgtgattcctagccacctcaaggtatgcctctccccggaaactgtggccttttctggcacacatgatctccacgatttcaacatataaatagcttttgataatggcaatattaatcaaatttattttacttctttcttgtaacatctctcttgtaatcccttattccttctagctatttttcataaaaaaccaagcaactgcttatcaacacacaaacactaaatcaaaatggctgttactaatgtcgctgaacttaacgcactcgtagagcgtgtaaaaaaagcccagcgtgaatatgccagtttcactcaagagcaagtagacaaaatcttccgcgccgccgctctggctgctgcagatgctcgaatcccactcgcgaaaatggccgttgccgaatccggcatgggtatcgtcgaagataaagtgatcaaaaaccactttgcttctgaatatatctacaacgcctataaagatgaaaaaacctgtggtgttctgtctgaagacgacacttttggtaccatcactatcgctgaaccaatcggtattatttgcggtatcgttccgaccactaacccgacttcaactgctatcttcaaatcgctgatcagtctgaagacccgtaacgccattatcttctccccgcacccgcgtgcaaaagatgccaccaacaaagcggctgatatcgttctgcaggctgctatcgctgccggtgctccgaaagatctgatcggctggatcgatcaaccttctgttgaactgtctaacgcactgatgcaccacccagacatcaacctgatcctcgcgactggtggtccgggcatggttaaagccgcatacagctccggtaaaccagctatcggtgtaggcgcgggcaacactccagttgttatcgatgaaactgctgatatcaaacgtgcagttgcatctgtactgatgtccaaaaccttcgacaacggcgtaatctgtgcttctgaacagtctgttgttgttgttgactctgtttatgacgctgtacgtgaacgttttgcaacccacggcggctatctgttgcagggtaaagagctgaaagctgttcaggatgttatcctgaaaaacggtgcgctgaacgcggctatcgttggtcagccagcctataaaattgctgaactggcaggcttctctgtaccagaaaacaccaagattctgatcggtgaagtgaccgttgttgatgaaagcgaaccgttcgcacatgaaaaactgtccccgactctggcaatgtaccgcgctaaagatttcgaagacgcggtagaaaaagcagagaaactggttgctatgggcggtatcggtcatacctcttgcctgtacactgaccaggataaccaaccggctcgcgtttcttacttcggtcagaaaatgaaaacggcgcgtatcctgattaacaccccagcgtctcagggtggtatcggtgacctgtataacttcaaactcgcaccttccctgactctgggttgtggttcttggggtggtaactccatctctgaaaacgttggtccgaaacacctgatcaacaagaaaaccgttgctaagcgagctgaaaacatgttgtggcacaaacttccgaaatctatctacttccgccgtggctccctgccaatcgcgctggatgaagtgattactgatggccacaaacgtgcgctcatcgtgactgaccgcttcctgttcaacaatggttatgctgatcagatcacttccgtactgaaagcagcaggcgttgaaactgaagtcttcttcgaagtagaagcggacccgaccctgagcatcgttcgtaaaggtgcagaactggcaaactccttcaaaccagacgtgattatcgcgctgggtggtggttccccgatggacgccgcgaagatcatgtgggttatgtacgaacatccggaaactcacttcgaagagctggcgctgcgctttatggatatccgtaaacgtatctacaagttcccgaaaatgggcgtgaaagcgaaaatgatcgctgtcaccaccacttctggtacaggttctgaagtcactccgtttgcggttgtaactgacgacgctactggtcagaaatatccgctggcagactatgcgctgactccggatatggcgattgtcgacgccaacctggttatggacatgccgaagtccctgtgtgctttcggtggtctggacgcagtaactcacgccatggaagcttatgtttctgtactggcatctgagttctctgatggtcaggctctgcaggcactgaaactgctgaaagaatatctgccagcgtcctaccacgaagggtctaaaaatccggtagcgcgtgaacgtgttcacagtgcagcgactatcgcgggtatcgcgtttgcgaacgccttcctgggtgtatgtcactcaatggcgcacaaactgggttcccagttccatattccgcacggtctggcaaacgccctgctgatttgtaacgttattcgctacaatgcgaacgacaacccgaccaagcagactgcattcagccagtatgaccgtccgcaggctcgccgtcgttatgctgaaattgccgaccacttgggtctgagcgcaccgggcgaccgtactgctgctaagatcgagaaactgctggcatggctggaaacgctgaaagctgaactgggtattccgaaatctatccgtgaagctggcgttcaggaagcagacttcctggcgaacgtggataaactgtctgaagatgcattcgatgaccagtgcaccggcgctaacccgcgttacccgctgatctccgagctgaaacagattctgctggatacctactacggtcgtgattatgtagaaggtgaaactgcagcgaagaaagaagctgctccggctaaagctgagaaaaaagcgaaaaaatccgcttaagtcgagagcttttgattaagccttctagtccaaaaaacacgtttttttgtcatttatttcattttcttagaatagtttagtttattcattttatagtcacgaatgttttatgattctatatagggttgcaaacaagcatttttcattttatgttaaaacaatttcaggtttaccttttattctgcttgtggtgacgcgtgtatccgcccgctcttttggtcacccatgtatttaattgcataaataattcttaaaagtggagctagtctatttctatttacatacctctcatttctcatttcctcctaatgtgtcaatgatcatattcttaactggaccgatcttattcgtcagattcaaaccaaaagttcttagggctaccacaggaggaaaattagtgtgatataatttaaataatttatccgccattcctaatagaacgttgttcgacggatatctttctgcccaaaagggttctaagctcaatgaagagccaatgtctaaacctcgttacattgaaaatacagtaaatggttccaccattattatgttggtcttgtttagtatggccgatcggcgtgtgttttgtttgcaccttttatatagtagaagaatatttgtcttaattcttattagtactgcaacctaaccactaattatcaacaattattggattatataaaggaggtaaattgccggattaaaatcaaatatcattcatcaacaagtattcatattgtcggcatatttttacatgcggtgtaagtatttggatcgtattcttatagtgtcaatacctcgaagcagcgtttcaagtaccagacgtatgtaggaactttttaacgtcgagtccgtaagatttgatcagtattaaaaaaatctagataaatgagtggtacaaataaaaacatcattaaaaatcgttaaataaaaaagtatgaagatcatctattaaagtattagtagccattagccttaaaaaaatcagtgctagtttaagtataatctcgggcgcgccggccgaggcggttaagcggattttttcgcttttttctcagctttagccggagcagcttctttcttcgctgcagtttcaccttctacataatcacgaccgtagtaggtatccagcagaatctgtttcagctcggagatcagcgggtaacgcgggttagcgccggtgcactggtcatcgaatgcatcttcagacagtttatccacgttcgccaggaagtctgcttcctgaacgccagcttcacggatagatttcggaatacccagttcagctttcagcgtttccagccatgccagcagtttctcgatcttagcagcagtacggtcgcccggtgcgctcagacccaagtggtcggcaatttcagcataacgacggcgagcctgcggacggtcatactggctgaatgcagtctgcttggtcgggttgtcgttcgcattgtagcgaataacgttacaaatcagcagggcgtttgccagaccgtgcggaatatggaactgggaacccagtttgtgcgccattgagtgacatacacccaggaaggcgttcgcaaacgcgatacccgcgatagtcgctgcactgtgaacacgttcacgcgctaccggatttttagacccttcgtggtaggacgctggcagatattctttcagcagtttcagtgcctgcagagcctgaccatcagagaactcagatgccagtacagaaacataagcttccatggcgtgagttactgcgtccagaccaccgaaagcacacagggacttcggcatgtccataaccaggttggcgtcgacaatcgccatatccggagtcagcgcatagtctgccagcggatatttctgaccagtagcgtcgtcagttacaaccgcaaacggagtgacttcagaacctgtaccagaagtggtggtgacagcgatcattttcgctttcacgcccattttcgggaacttgtagatacgtttacggatatccataaagcgcagcgccagctcttcgaagtgagtttccggatgttcgtacataacccacatgatcttcgcggcgtccatcggggaaccaccacccagcgcgataatcacgtctggtttgaaggagtttgccagttctgcacctttacgaacgatgctcagggtcgggtccgcttctacttcgaagaagacttcagtttcaacgcctgctgctttcagtacggaagtgatctgatcagcataaccattgttgaacaggaagcggtcagtcacgatgagcgcacgtttgtggccatcagtaatcacttcatccagcgcgattggcagggagccacggcggaagtagatagatttcggaagtttgtgccacaacatgttttcagctcgcttagcaacggttttcttgttgatcaggtgtttcggaccaacgttttcagagatggagttaccaccccaagaaccacaacccagagtcagggaaggtgcgagtttgaagttatacaggtcaccgataccaccctgagacgctggggtgttaatcaggatacgcgccgttttcattttctgaccgaagtaagaaacgcgagccggttggttatcctggtcagtgtacaggcaagaggtatgaccgataccgcccatagcaaccagtttctctgctttttctaccgcgtcttcgaaatctttagcgcggtacattgccagagtcggggacagtttttcatgtgcgaacggttcgctttcatcaacaacggtcacttcaccgatcagaatcttggtgttttctggtacagagaagcctgccagttcagcaattttataggctggctgaccaacgatagccgcgttcagcgcaccgtttttcaggataacatcctgaacagctttcagctctttaccctgcaacagatagccgccgtgggttgcaaaacgttcacgtacagcgtcataaacagagtcaacaacaacaacagactgttcagaagcacagattacgccgttgtcgaaggttttggacatcagtacagatgcaactgcacgtttgatatcagcagtttcatcgataacaactggagtgttgcccgcgcctacaccgatagctggtttaccggagctgtatgcggctttaaccatgcccggaccaccagtcgcgaggatcaggttgatgtctgggtggtgcatcagtgcgttagacagttcaacagaaggttgatcgatccagccgatcagatctttcggagcaccggcagcgatagcagcctgcagaacgatatcagccgctttgttggtggcatcttttgcacgcgggtgcggggagaagataatggcgttacgggtcttcagactgatcagcgatttgaagatagcagttgaagtcgggttagtggtcggaacgataccgcaaataataccgattggttcagcgatagtgatggtaccaaaagtgtcgtcttcagacagaacaccacaggttttttcatctttataggcgttgtagatatattcagaagcaaagtggtttttgatcactttatcttcgacgatacccatgccggattcggcaacggccattttcgcgagtgggattcgagcatctgcagcagccagagcggcggcgcggaagattttgtctacttgctcttgagtgaaactggcatattcacgctgggctttttttacacgctctacgagtgcgttaagttcagcgacattagtaacagccataattcttaattaactttgatatgattttgtttcagattttttatataaaagctttcccaaatagtgctaaagtgaacttagattttttggtacctgtttcgaaattaaaaatagaaaaatttctctccctatattgttattcttacttcaaatttgtttatcgtttatttactaggcgagacttgagtagacgacaatccaaatagaattaacagattttattggtagaaagcaataatattctttagatggttgagaataaagaagtaaaaaaaccagtaaagagaaaaagaaaaggaagaaaattaaagaaaaaggatgattacacaagaagataataaaaaaactcctttattaagagcggaagaatttaataatgaagatgggaataagcaaaacaaaaacaaagaagggaaaaaaaataaaaaatcgtatttatttatttaaaaaatcatgttgatgacgacaatggaaaaaaaaaaccgatttcactttctcatccttatatttttcaaaggttgatgcaagtcgatctcaaatcggataacgctgccaactgggaaattccgcaattccgcaagaaaaaaaaaaatgtgaaaacgtgattgcattttttacaggtcctaaaggatttagcccacatatcaagagggtggcagtaattgcactgattaagcattcgtcagcattaggcgaatgtgtgcatgaatattgccagtgtgctcgatattagagagtacattgaagaatattgtaccggattatgtacaataactttgttaatgagatattaattttcttttttactagccgctatcccatgcacgatgctaaatttcaagaagaaactgagatttaaaaaattagtggaagctgataaaacggactataatggtgtatggattgaggaatctcgacatgtttttccatcgttttcaacgatgactgtaacccgtagattgaaccaggcatgccaaagttagttagatcagggtaaaaattatagatgaggtttaattaaacaagcacgcagcacgctgtatttacgtatttaattttatatatttgtgcatacactactagggaagacttgaaaaaaacctaggaaatgaaaaaacgacacaggaagtcccgtatttactattttttccttccttttgatggggcagggcggaaatagaggataggataagcctactgcttagctgtttccgtctctacttcggtagttgtctcaattgtcgtttcagtattacctttagagccgctagacgatggttgagctatttgttgagggaaaactaagttcatgtaacacacgcataacccgattaaactcatgaatagcttgattgcaggaggctggtccattggagatggtgccttattttccttataggcaacgatgatgtcttcgtcggtgttcaggtagtagtgtacactctgaatcagggagaaccaggcaatgaacttgttcctcaagaaaatagcggccataggcatggattggttaaccacaccagatatgcttggtgtggcagaatatagtccttttggtggcgcaattttcttgtacctgtggtagaaagggagcggttgaactgttagtatatattggcaatatcagcaaatttgaaagaaaattgtcggtgaaaaacatacgaaacacaaaggtcgggccttgcaacgttattcaaagtcattgtttagttgaggaggtagcagcggagtatatgtattccttttttttgcctatggatgttgtaccatgcccattctgctcaagcttttgttaaaattatttttcagtattttttcttccatgttgcgcgttacgagaacagaagcgacagataaccgcaatcatacaactagcgctactgcggggtgtaaaaagcacaagaactaagccaagatcacaacagttatcgataaaatagcagtgtttgcatggccattgagaaggacaacattggcgtgcgcgccaatgttgtctcaccatgtagctccaaacgagttgtaagagacggaccgctcacgcttccgaagcggtcagaaaacgcttcccagtatgcagttgacctacattcaacctgcaaatattgctttgcttcaagaaatgattacacagacgtctattttcttctacataatgcacgaaacttgggcatttagtcatgtagccgcctagcgagcctgggtgccgtcctatctcctttgttcgtgcaaagagacaggaacacacactgcgttctcttgcggccggtctggcggactcaggggtgcggcgtttgcttaaccggagggaataataaaatcggggtgacgcaagtatgaagtcatgtgtgcttagcaattacgtagagggattagaaataatagtgtgcggttatcggaaccggctcttgttcccgtttagagcaacccaggtgcaggcgtactttaaagtattttctttcttttttttcctgctacttacgctaggagctgccgcagctgcaaagccgacgtcggagaggcaggtgatcttcggctcggccgacaaatcccctggatatcattggcctgtcgaggtatcggccgcgtggaactaccgggaattactatgcaaaacaattggaaatctggtaggaaaaccttgttctagaacttggcgattgctgacaaagaagaaaagggcctattgttgctgcctcttttgttgttcttcctcgtattgtcttgccggtgttctttgtgtcttttgtgtgtaggttcttactattatagtgctctttgctattatattttcttcgttttcactttgcgtaatgtaacggtcttaaacaaagttttttttttttcgctcttgcattttccttttctgctctatcttatttgctaattgtagtttcagaagttttacttaaatatagcactattttccagttttaatgtttcttctcattgctttcttttataattttcgcatataattatacatttacggtgtcttaactctccctcttcacccctcattattccagaaaatactaatacttcttcacacaaaagaacgcagttagacaatcaacaatgaatcctaaatcctctacacctaagattccaagacccaagaacgcatttattctgttcagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtgtggaaataccccataattcaaacatttctaaaattattggtacgaagtggaagggcttacaaccggaagataaggcacactgggaaaatctagcggagaaggagaaactagaacatgaaaggaagtatcctgaatacaaatacaagccggtaagaaagtctaagaagaagcaactacttttgaaggaaatcgagcaacagcagcagcaacaacagaaagaacagcagcagcagaaacagtcacaaccgcaattacaacagccctttaacaacaatatagttcttatgaaaagagcacattctctttcaccatcttcctcggtgtcaagctcgaacagctatcagttccaattgaacaatgatcttaagaggttgcctattccttctgttaatacttctaactatatggtctccagatctttaagtggactacctttgacgcatgataagacggcaagagacctaccacagctgtcatctcaactaaattctattccatattactcagctccacacgacccttcaacgagacatcattacctcaacgtcgctcaagctcaaccaagggctaactcgacccctcaattgccctttatttcatccattatcaacaacagcagtcaaacaccggtaactacaactaccacatccacaacaactgcgacatcttctcctgggaaattctcctcttctccgaactcctctgtactggagaacaacagattaaacagtatcaacaattcaaatcaatatttacctccccctctattaccttctctgcaagattttcaactggatcagtaccagcagctaaagcagatgggaccaacttatattgtcaaaccactgtctcacaccaggaacaatctattgtccacaactacccctacgcatcatcacattcctcatataccaaaccaaaacattcctctacatcaaattataaactcaagcaacactgaggtcaccgctaaaactagcctagtttctccgaaatgattttttttttccatttcttctttccgttatattatattatactatattccctttaactaaaaatttatgcatttggctcctgtttaataaaagtttaaatc

These strains were grown in YPD containing 50 g/L glucose underanaerobic and microaerobic conditions, and formate was measured over 142hours. As shown in FIG. 14, at the end of 142 hours, strains containingthe heterologous PFL and AADH made more formate than the wildtype strainM139.

Example 6 Expression of PFL and AADH and Detection of Ethanol

The purpose of this Example is to determine whether formate productioncan confer anaerobic growth on fdh, gpd, and/or fps deletion strains.Yeast strains containing an fdh1Δfdh2Δgpd1Δgpd2Δ genetic background(M2025) were transformed with vectors expressing PflA/B cassettes fromC. cellulolyticum (TB274) and E. coli (TB275). Each of these strainsalso contained a second construct expressing the E. coli AdhE. YPDmedium was prepared and added to hungate tubes, oxygen was purged withnitrogen, and the tubes were autoclaved for 20 minutes. A pre-culture ofTB274 and TB275 was prepared overnight in YPD medium containingantibiotics which select for maintenance of both plasmids. A pre-cultureof M1901, the parent strain of M2025, and M2025 itself were prepared inYPD and included as positive and negative controls, respectively. Astrain referred to as TB267 was created which contains only thebifunctional ADH plasmid. This strain was prepared in YPD plusantibiotic to select for the plasmid. This strain controls for thepotential effect of ADH or other electron acceptors that may be presentin YPD medium.

All strains were inoculated to final OD's of about 0.05 or below. The ODof each culture was measured at 0, 24, 48, and 72 hours (FIG. 15), andsamples were prepared and submitted for HPLC determination of metabolitelevels. As expected, the M1901 strain grew fairly quickly, consuming allthe sugar substrate by 24 hours. The M2025 and TB267 strains, which areunable to make glycerol, did not show a significant increase in ODduring this experiment. The TB274 and TB275 strains, which express bothPFL and ADH, were able to grow after a 24 hour lag time. See FIG. 15.These data indicate that the introduced metabolic pathway in TB274 andTB275 does not block cellular growth.

The production of glycerol in these strains is shown in FIG. 16. Theanaerobic growth of M1901 was accompanied by glycerol production asexpected. A trace amount of glycerol was observed in the PflA/Bcontaining strains TB274 and TB275, but this was at or below the levelof the negative controls which did not grow. See FIG. 16. These data, inconjunction with the OD data, indicate that the expression of PflA/B andAdhE allowed for anaerobic growth of M2025 without associated glycerolproduction.

The production of ethanol and glucose concentration are shown in FIGS.17 and 18, respectively. Both M1901 and TB274 consumed all the sugar,but about 5 g/L glucose remained in the TB275 fermentation. See FIG. 18.TB274 had an 11% increase in ethanol yield in comparison to M1901. SeeFIG. 17. The increase in yield was higher than expected and likely camepartially at the expense of biomass, although this was not determined.

Strains containing PFL and AADH were compared to other strainsengineered to express AADH. A description of these strains appears inTable 4.

TABLE 4 Genetic Backgrounds for PFL Expression Strain Name GeneticBackground M139 wt control M2085 gdp1Δ gpd2Δ fdh1Δ fdh2Δ M2158 gdp1Δgpd2Δ fdh1Δ fdh2Δ + AADH M2182 gdp1Δ gpd2Δ fdh1Δ fdh2Δ + AADH and PFL

These five strains were tested in nitrogen purged bottles. As shown inFIG. 19, strain M2182 which has both PFL and AADH expression cassettesin a gdp1Δ gpd2Δ fdh1Δ fdh2Δ background, had a faster growth rate andreached a higher OD than M2158, which contains only the bifunctional ADHactivity and did not appear to grow at all. See FIG. 19. These data showthat a strain expressing both PFL and AADH activities grows better thanthe other engineered functionalities. Similar improvements over theseother engineered functionalities are also observed in corn mashfermentations (see Example 7 below).

Example 7 Production of Ethanol from Corn Mash

The purpose of this experiment was to determine whether PFLs cloned fromthe organisms listed below in Table 5 could provide for increasedethanol yield when used in fermentation of 20% corn mash. A total ofnine PFLs have been tested for function in yeast. Of these, only the C.cellulolyticum PFL had no positive effect on growth of glycerolsynthesis mutants in corn mash fermentations. Additionally, no formatewas observed in formate assays when using a strain containing C.cellulolyticum PFL. This strain was not tested for performance on cornmash fermentation.

Eight PFLs were tested for functionality in strain M2158 which has theE. coli AADH integrated on the chromosome of a gpd1Δ gpd2Δ fdh1Δ fdh2Δbackground (M2085) or M2275, which is identical to M2158 except that italso has the gpd2Δ::GPD1 glycerol reduction mutation. Two separate cornmash fermentation experiments were performed using 20% solids in abaffled shake flask using the strains listed in Table 5. Performance ofthe strains was evaluated by HPLC analysis of metabolites.

TABLE 5 Genetic Backgrounds for Corn Mash Fermentations Strain NameGenetic Background M139 wt control M2085 gpd1Δ gpd2Δ fdh1Δ fdh2Δ M2158integrated AADH M2180 M2158 + B. lichenformis PFL M2181 M2158 + L.planatarum PFL M2182 M2158 + B. adolescentis PFL M2183 M2158 + S.thermophilus PFL M2184 M2158 + E. coli PFL M2321 M2158 + L. casei PFLM2322 M2158 + C. reinhardtii PFL M2323 M2158 + Piromyces PFL M2324M2158 + E. coli PFL M2275 M2158 + gpdΔ::GPD1 M2326 M2275 + B.adolescentis PFL

As shown in FIGS. 21 and 22, the addition of PFL improves the ethanolyield of strains containing only AADH. FIGS. 23 and 24 show that thesestrains do not make glycerol as expected. The approximately 4 g/Lglycerol observed is already in the industrial mash substrate used forthis experiment. FIG. 25 demonstrates a 9% increase in ethanol yieldwith strain M2326, which is a glycerol reduction strain.

Example 8

The following example demonstrates the creation of the Saccharomycescerevisiae strain M3625. The genotype of strain M3625 is: Δgpd2:: B.adolescentis pflA/pflB/adhE Δfdh1 Δfdh2::B. adolescentis pflA/pflB/adhEfcy1Δ::S. fibuligera glucoamylase (glu-0111-CO). Strain M2390 isreferred to as Ethanol Red (new) from LaSaffre(pahc.com/Phibro/Performance-Products/Catalog/23/Ethanol-Red.html).

The genetic modification techniques utilized to develop Saccharomycescerevisiae strain M3625 relied upon directed integration to insert thegenes for Bifidobacterium adolescentis pflA, pflB, AdhE and S.fibuligera glu-0111-CO at specific and known sites within the yeastchromosome. The directed integration approach creates transgenic strainswith integration events that are stable and easy to characterize.Chromosomal integration, by its very nature, reduces the probability ofany mobilization of the heterologous DNA and enhances strain stabilityrelative to other approaches.

The MX cassettes are the most commonly used engineering tool when aninsertion or deletion of a genetic element is desired at a givenchromosomal loci (Wach A, et al., Yeast 10(13):1793-1808 (1994)). Arecyclable MX cassette contains one or more markers which enable bothdominant and negative selection (Goldstein, A. L. and McCusker, J. H.,Yeast 15:1541-1553 (1999); Ito-Harashima, S. and McCusker, J. H., Yeast21:53-61 (2004)). The dominant marker enables selection for themodification and the counter selectable marker enables subsequentremoval of the marker system via Cre-Lox mediated recombination(Güldener, Ulrich, et al., Nucleic Acids Research (1996) 24(13)2519-2524) or recombination between duplicated homologous regionsflanking the cassette. Since the markers are removed, they can be reusedduring subsequent engineering steps and ensures no undesirable foreigngenetic material remains in the strain.

To create stable homozygous integrations in M3625, two new HSV-thymidinekinase (TDK) based MX cassettes were developed. Expression of thymidinekinase in S. cerevisiae results in sensitivity to the compoundfluoro-deoxyuracil (FUDR). The cellular toxicity of FUDR is dependent onthe presence of two enzymes involved in pyrimidine metabolism: thymidinekinase (Tdk) and thymidilate synthetase (ThyA). Tdk converts FUDR tofluoro-dUMP (F-dUMP) which is a covalent inhibitor of ThyA and the basisfor counter selection in a variety of eukaryotic organisms (Czako, M.,and L. Marton, (1994) Plant Physiol 104:1067-1071; Gardiner, D. M., andB. J. Howlett, (2004) Curr Genet 45:249-255; Khang, C. H., et al.,(2005) Fungal Genet Biol 42:483-492; Szybalski, W. (1992) Bioessays14:495-500).

The HSV-TDK expression cassette was independently fused to two commonlyused dominant selectable markers which confer resistance to the drugsG418 (Kan) or nourseothricin (Nat) (Goldstein, A. L. and McCusker, J.H., Yeast 15:1541-1553 (1999)). Transformation of both double expressioncassettes, referred to as KT-MX and NT-MX, enables positive selectionfor integration into both chromosomes as illustrated in FIG. 28A. Thetransformed deletion assembly contains four PCR products, a 5′flank(p1)which is homologous upstream of the target site, KT-MX cassette(p2),NT-MX cassette(p3), and a 3′ flank(p4) homologous downstream of thetarget site. Each component is amplified individually using primerswhich create homologous overlapping extensions of each PCR product. SeeTables 7 and 8. The bent dashed lines in FIG. 28A represent homologybetween the KT/NT-MX cassettes and the 5′ flank and the bent solid linesrepresent homology with the 3′ flank. For each round of engineering, PCRamplicons of upstream and downstream regions flanking the target siteare designed to contain homologous tails for both the KT-MX and NT-MXcassettes. Both the flanks and the markers are transformed followed byselection on YPD medium containing both G418 and Nat. See FIG. 28B. FIG.28B shows a schematic of the chromosome after replacement of the targetsite with KT-MX and NT-MX.

After each engineering step taken in the construction of M3625, allmarkers are subsequently deleted and/or replaced with a desiredexpression cassette (Mascoma Assembly) resulting in a strain free ofantibiotic markers (FIG. 29). FIG. 29 demonstrates that the transformedMascoma Assembly contains a quantity of PCR products which is dependenton the desired engineering event (pX), a 5′flank(p1) which homologousupstream of the target site and a 3′ flank (p4) homologous downstream ofthe target site. Each component is amplified individually using primerswhich create homologous overlapping extensions. The overlapping bentlines in FIG. 29 represent homology at the end of those PCR products.FIG. 29B shows a schematic of a chromosome following selection on FUDRand replacement of genetic markers with the Mascoma assembly.Confirmation of marker removal was evaluated by southern blot, PCR anddilution plating onto selective medium as described below.

Four loci were modified during the construction of M3625. Theintegration procedure strategy described above was used at the FDH1,GPD1 and GPD2 loci using the Mascoma Assemblies listed in Table 6.Detailed molecular maps depicting the components of each MascomaAssembly are provided in FIGS. 30-37.

TABLE 6 Genetic modifications contained in M3625. Target Locus LocusModification Cassette ID Cassette Description FDH1 Clean Deletion MA0370Clean Deletion of FDH1 FDH2 Replaced with MA0280 2 copies of pflA/Bexpression cassette and 4 copies of adhE GPD2 Replaced with MA0289 2copies of pflA/B expression cassette and 4 copies of adhE FCY1 Replacedwith MA0317 Four copies of expression cassette Glucoamylase

TABLE 7 Primers used for the creation of strain M3625. PCR Product 1;PCR Product 2; PCR Product 3; Target Locus Primer Pair Primer PairPrimer Pair GPD1 GPD1 5′ Flank; pAGTEF-kan/nat- GPD1 3′ Flank;X11824/X15546 pHXT2-TDK; X15547/X11829 X15380/X15382 GPD2 GPD2 5′ Flank;pAGTEF-kan/nat- GPD2 3′ flank; X11816/X15548 pHXT2-TDK; X15549/X11821X15380/X15382 FDH2 FDH2 5′ Flank; pAGTEF-kan/nat- FDH2 3′ flank;X16096/X15554 pHXT2-TDK; X15555/X11845 X15380/X15382 FDH1 FHD1 5′ Flank;pAGTEF-kan/nat- FDH1 3′ flank; X15559/X15550 pHXT2-TDK; X15552/X15553X15380/X15382

TABLE 8 Sequences of Primers used for creation of strain M3625 SEQ IDPrimer Sequence NO X11824 aagcctacaggcgcaagataacacatcac 110 X15546ggacgaggcaagctaaacagatctctagacctactttatattatcaatatttgtgtttg 111 X15380taggtctagagatctgtttagcttgc 112 X15382 gagactacatgatagtccaaaga 113 X15547ccgtttcttttctttggactatcatgtagtctcatttattggagaaagataacatatca 114 X11829ctcagcattgatcttagcagattcaggatctaggt 115 X11816gcagtcatcaggatcgtaggagataagca 116 X15548ggacgaggcaagctaaacagatctctagacctatgataaggaaggggagcg 117 aaggaaaa X15549ccgtttcttttctttggactatcatgtagtctcctctgatctttcctgttgcctctttt 118 X11821tcacaagagtgtgcagaaataggaggtgga 119 X16096 catggtgcttagcagcagatgaaagtgtca120 X15554 ggacgaggcaagctaaacagatctctagacctaattaattttcagctgttatttcgatt121 X15555 ccgtttcttttctttggactatcatgtagtctcgagtgattatgagtatttgtgagcag122 X11845 ttacttgtgaaactgtctccgctatgtcag 123 X15559ggaaggcaccgatactagaactccg 124 X15550gggacgaggcaagctaaacagatctctagacctaattaattttcagctgttattttgat 125 X15552ccgtttcttttctttggactatcatgtagtctcgagtgattatgagtatttgtgagcag 126 X15553accagcgtctggtggacaaacggccttcaac 127

Genotyping and Sequencing of MA0370

To confirm that FDH1 was deleted after insertion of MA370, PCR productswere amplified from M2390 and M3625 genomic DNA using primers X17826 andX16944. The expected results are listed in Table 10 and the sequences ofthe primers used are listed in Table 11. A molecular map depicting theMA0370 integration site is shown in FIG. 30. The molecular map depictsthe location of flanks used to remove the KT-MX and NT-MX markers andthe position of primers used for genotyping. See FIG. 30 (5′ flank, S.cerevisiae FDH1 upstream flanking region; 3′ flank—S. cerevisiae FDH1downstream flanking region. Region AA—amplified and sequencedchromosomal DNA region). Primer pair X15556/X15871 was used for the FDH15′ Flank and primer pair X15870/X15553 was used for the FHD1 3′ Flank tocreate the assembly shown in FIG. 30. Sequences for the primers forassembly used are found in Table 9. An agarose gel image showing PCRproducts used to determine genotype is shown in FIG. 31 (lane 1: 1 KBladder; lane 2: M2390 (X17826/X16944); lane 3: M3625 (X17826/X16944))(see Table 11).

TABLE 9 Primers used to create the MA0370 integration site. SEQ IDPrimer Sequence NO X15556 ccactcgaggataggcttgaaaga 128 X15870ctaatcaaatcaaaataacagctgaaaattaatgagtgat 129 tatgagtatttgtgagcag X15871aaaacttctgctcacaaatactcataatcactcattaatt 130 ttcagctgttattttgatt X15553accagcgtctggtggacaaacggccttcaac 127

In order to determine the exact DNA sequence of the M3625 MA0370 site,region AA was amplified from genomic DNA of M3625 strain in 5independent PCR reactions. All PCR products were purified and sequencedby the Sanger method at the Dartmouth College Sequencing facility.

TABLE 10 Primers and summary of results of MA0370 genotyping. ExpectedCorrect Size Lane Template DNA Primers size(bp) Observed 1 1KB ladderN/A N/A N/A 2 M2390 X17826/X16944 4386 bp yes 3 M3625 X17826/X169443237   yes

TABLE 11 Sequence of primers used for MA0370 genotyping. Primer SequenceSEQ ID NO X17826 tcgctaacgatcaagaggaactg 152 X16944tacacgtgcatttggacctatc 153

Genotyping and Sequencing of MA0280

To confirm that MA280 was inserted at the FDH2 site, PCR products wereamplified from M3625 genomic DNA. The primers and expected genotypingresults are listed in Table 13. Sequences of the primers used forgenotyping and sequencing MA0280 are listed in Table 14. A molecular mapdepicting the MA0280 integration site is shown in FIG. 32. The molecularmap depicts the location of flanks used to replace the KT-MX and NT-MXmarkers and insert the MA0280 expression cassette. The position ofprimers used for genotyping for genotyping are indicated on the map. SeeFIG. 32 (Feature description on map of MA0280 site of the M3625 strain;FDH2 5′ flank—S. cerevisiae FDH2 upstream flanking region; PFK1p—S.cerevisiae PFK1 gene promoter; ADHE—Bifidobacterium adolescentis ADHEcoding gene; HXT2t—S. cerevisiae HXT2 gene terminator; ENO1p—S.cerevisiae ENO1 gene promoter; PFLB—Bifidobacterium adolescentis PFLBcoding gene; ENO1t—S. cerevisiae ENOL gene terminator; ADH1p—S.cerevisiae ADH1 gene promoter; PFLA—Bifidobacterium adolescentis PFLAcoding gene; PDC1t—S. cerevisiae PDC1 gene terminator; FBA1t—S.cerevisiae FBA1 gene terminator; TPI1p—S. cerevisiae TPI1 gene promoter;FDH2 3′ flank—S. cerevisiae FDH2 downstream flanking region. RegionsBA-BE, amplified and sequenced chromosomal DNA regions). Primer pairX16096/X17243 was used for the FDH2 5′ Flank, primer pair X16738/X16620was used for pPFK1-ADH-HXT2, primer pair X16621/X13208 was used forpENO1-PFL-ENO1t, primer pair X13209/X17242 was used for pADH1-PFL-PDC1t,primer pair X17241/X16744 was used for pTPI-ADH-FBA1trc, and primer pairX17244/X11845 was used for the FDH2 5′ Flank to create the assemblyshown in FIG. 32. Sequences for the primers used to create the assemblyshown in FIG. 32 are found in Table 12. An agarose gel image showing PCRproducts used to genotype and sequence the MA0280 site is shown in FIG.33 (lane 1: 1 KB ladder; lane 2: M3625 (17413/15810); lane 3: M3625(17834/14554); lane 4: M3625 (16291/15229); lane 5: M3625 (16503/11317);lane 6: (16241/16946) lane 7: 1 KB ladder) (see Table 14).

TABLE 12 Primers used to create the MA0280 integration site. SEQ IDPrimer Sequence NO X16096 catggtgcttagcagcagatgaaagtgtca 120 X17243tagttagatcagggtaaaaattatagatgaggtattaatt 131 ttcagctgttatttcgatt X16738ctaatcaaatcgaaataacagctgaaaattaatacctcat 132 ctataatttttaccctgat X16620tcggatcagtagataacccgcctagaagactaggttac 133 attgaaaatacagtaaatggt X16621tggtggaaccatttactgtattttcaatgtaacctagtc 134 ttctaggcgggttatctact X13208ccgaaatattccacggtttagaaaaaaatcggaggttt 135 agacattggctcttcattgag X13209aagctcaatgaagagccaatgtctaaacctccgatttttt 136 tctaaaccgtggaatattt X17242acatcatcttttaacttgaatttattctctagctttcaa 137 tcattggagcaatcatttta X17241gtccatgtaaaatgattgctccaatgattgaaagctagag 138 aataaattcaagttaaaag X16744aaaaacttctgctcacaaatactcataatcactcctac 139 ttattcccttcgagattatatc X17244gttcctagatataatctcgaagggaataagtaggagtg 140 attatgagtatttgtgagcag X11845ttacttgtgaaactgtctccgctatgtcag 141

In order to determine exact DNA sequence of the M3625 MA0280 site,regions BA-BE were amplified from genomic DNA of M3625 strain in 5independent PCR reactions. All PCR products were purified and sequencedby the Sanger method at the Dartmouth College Sequencing facility.

TABLE 13 Primers and summary of results of MA0280 genotyping. ExpectedCorrect Size Lane Template DNA Primers size(bp) Observed 1 1 KB ladderN/A N/A N/A 2 M3625 17413/15810 5567 Yes 3 M3625 17834/14554 3686 Yes 4M3625 16291/15229 2569 Yes 5 M3625 16503/11317 4352 yes 6 M362516241/16946 2478 yes 7 1KB ladder N/A N/A N/A

TABLE 14 Sequence of primers used for genotyping MA0280. Primer SequenceSEQ ID NO 17413 ggattcttcgagagctaaga 154 15810gacttgcagggtaggctagctagaatt 155 17834 gctgcttcgaggtattgaca 156 14554ggctcttcattgagcttagaaccc 157 16291 aactggaccgatcttattcgt 158 15229agtccactgcggagtcatttcaaag 159 16503 ctgccagcgaattcgactctgcaat 160 11317cagtcgctgtagtgagcgacagggtagtaa 161 16241 ctttgcattagcatgcgta 162 16946taggtcgagaccagaatgcatgt 163

Genotyping and Sequencing of MA0289

To confirm that MA0289 was inserted at the GPD2 site, PCR products wereamplified from M3625 genomic DNA. The primers and expected genotypingresults are listed in Table 16. Sequences for the primers used forgenotyping MA0289 are listed in Table 17. A molecular map depicting theMA0289 integration site is shown in FIG. 34. The molecular map depictsthe location of flanks used to replace the KT-MX and NT-MX markers andinsert the MA0280 expression cassette. The position of primers used forgenotyping are indicated on the map. See FIG. 34 (Feature description onmap of MA0280 site of the M3625 strain; GPD2 5′ flank—S. cerevisiae GPD2upstream flanking region; ADHE—Bifidobacterium adolescentis ADHE codinggene; HXT2t—S. cerevisiae HXT2 gene terminator; PDC1t—S. cerevisiae PDC1gene terminator; PFLA—Bifidobacterium adolescentis PFLA coding gene;ADH1p—S. cerevisiae ADH1 gene promoter; ENO1t—S. cerevisiae ENOL geneterminator; PFLB—Bifidobacterium adolescentis PFLB coding gene; ENO1p—S.cerevisiae ENOL gene promoter; FBA1t—S. cerevisiae FBA1 gene terminator;TPI1p—S. cerevisiae TPI1 gene promoter; GPD2 3′ flank—S. cerevisiae GPD2downstream flanking region; Regions CA-CF—amplified and sequencedchromosomal DNA regions). Primer pair X15473/X17460 was used for theGPD2 5′ Flank, primer pair X17459/X17289 was used for ADH-HXT2, primerpair X17290/X13209 was used for pADH1-PFL-PDC1trc, primer pairX13208/X15735 was used for pENO1-PFL-ENO1trc, primer pair X15736/X17457was used for pTPI-ADH-FBA1trc, and primer pair X17458/X15476 was usedfor the GPD2 3′ Flank to create the assembly shown in FIG. 34. Sequencesfor the primers used to create the assembly shown in FIG. 34 are foundin Table 15. An agarose gel image showing PCR products used to genotypeand sequence the MA0280 site is shown in FIG. 35 (lane 1: 1 KB ladder;lane 2: M3625 (17413/15810); lane 3: M3625 (17834/14554); lane 4: M3625(16291/15229); lane 5: M3625 (16503/11317); lane 6: (16241/16946); lane7: 1 KB ladder).

TABLE 15 Primers used to create the MA0289 integration site. SEQ IDPrimer Sequence NO X15473 agtcatcaggatcgtaggagataagc 142 X17460agaagataatatttttatataattatattaatcctaatct 143 tcatgtagatctaattctt X17459cctttccttttccttcgctccccttccttatcaatggcaga 144 cgcaaagaagaaggaaga X17289gtccatgtaaaatgattgctccaatgattgaaagttacat 145 tgaaaatacagtaaatggt X17290tggtggaaccatttactgtattttcaatgtaactttcaat 146 cattggagcaatcatttta X13208ccgaaatattccacggtttagaaaaaaatcggaggtttag 135 acattggctcttcattgag X13209aagctcaatgaagagccaatgtctaaacctccgattttt 136 ttctaaaccgtggaatattt X15735catcttttaacttgaatttattctctagcctagtcttcta 147 ggcgggttatctactgat X15736agataacccgcctagaagactaggctagagaataaattcaa 148 gttaaaagatgatgttga X17457tgggggaaaaagaggcaacaggaaagatcagagctactt 149 attcccttcgagattatatc X17458gttcctagatataatctcgaagggaataagtagctctgat 150 ctttcctgttgcctcttttt X15476gtagatctgcccagaatgatgacgtt 151

In order to determine exact DNA sequence of the M3625 MA0289 site,regions CA-CF were amplified from genomic DNA of M3625 strain in 5independent PCR reactions. All PCR products were purified and sequencedby the Sanger method at the Dartmouth College Sequencing facility.

TABLE 16 Primers and summary of results of MA0289 genotyping. ExpectedCorrect Size Lane Template DNA Primers size(bp) Observed 1 1 KB ladderN/A N/A N/A 2 M3625 16939/16940 2477 Yes 3 M3625 16807/14567 3831 Yes 4M3625 17834/14557 2478 Yes 5 M3625 16640/14552 3978 yes 6 M362517586/16806 4789 yes 7 1KB ladder N/A N/A N/A

TABLE 17 Sequence of primers used for genotyping MA0289. Primer SequenceSEQ ID NO 16939 atgctgatgcatgtccacaaag 164 16940 ccttatcagtcaattgaggaaag165 16807 gcgatgagctaatcctgagccat 166 14567 tggttccaccattattatgttggt 16717834 gctgcttcgaggtattgaca 168 14557 ctaaaccgtggaatatttcggatat 169 16640cctcatcagctctggaacaacga 170 14552 gatccgagcttccactaggatagc 171 17586gcagtatgcaagtctcatgctg 172 16806 gaacttgcaggcaccgatcttca 173

Genotyping and Sequencing of MA0317

To confirm that MA0317 was inserted at the FCY1 site, PCR products wereamplified from M3625 genomic DNA. The primers and expected genotypingresults are listed in Table 19. Sequences for the primers used togenotype MA0317 are listed in Table 20. A molecular map depicting theMA0317 integration site is shown in FIG. 36. The molecular map depictsthe location of flanks used to replace the FCY1 gene with MA0317 and theposition or primers used for genotyping. See FIG. 36 (Featuredescription on the map of MA0371 site of M3625; FCY1 5′ flank—S.cerevisiae FCY1 upstream flanking region; ENO1p—S. cerevisiae ENOL genepromoter; AE9 —S. fibuligera glu 0111 coding gene; ENO1t—S. cerevisiaeENOL gene terminator; PDC1t—S. cerevisiae PDC1 terminator; ADH1p—S.cerevisiae ADH1 gene promoter; FCY1 3′ flank—S. cerevisiae FCY1downstream flanking region; Regions FA-FE, amplified and sequencedchromosomal DNA regions). Primer pair X18868/X18844 was used for the FCY5′ Flank, primer pair X18845/X15464 was used for pENO-AE9-ENO1t, primerpair X15465/X11750 was used for pADH1-AE9-PDC1t, and primer pairX15479/X18869 was used for the FCY 3′ Flank to create the assembly shownin FIG. 36. Sequences for the primers used to create the assembly shownin FIG. 36 are found in Table 18. An agarose gel image showing PCRproducts used to determine genotype is shown in FIG. 37 (lane 1: 1 KBladder; lane 2: M3625 (13092/17586); lane 3: M3625 (10871/14554); lane4: M3625 (16291/17887); lane 5: M3625 (16640/16509); lane 6: M3625(13246/13095); lane 7: 1 KB ladder).

TABLE 18 Primers used to create the MA0317 integration site. SEQ IDPrimer Sequence NO X18868 gccaaagtggattctcctactcaagctttgc 184 X18844tcggatcagtagataacccgcctagaagactagtagctat 185 gaaatttttaactctttaa X18845agccagcttaaagagttaaaaatttcatagctactagtctt 186 ctaggcgggttatctact X15464gtccatgtaaaatgattgctccaatgattgaaagaggttta 187 gacattggctcttcattg X15465ctaagctcaatgaagagccaatgtctaaacctctttcaatc 188 attggagcaatcatttta X11750ataaaattaaatacgtaaatacagcgtgctgcgtgctcg 189 atttttttctaaaccgtgga X15479agcacgcagcacgctgtatttacgta 190 X18869 agatcctgtggtagtgctgtctgaacagaa 191

In order to determine exact DNA sequence of the M3625 MA0317 site,regions FA-FE were amplified from genomic DNA of M3625 strain in 5independent PCR reactions. All PCR products were purified and sequencedby the Sanger method at the Dartmouth College Sequencing facility.

TABLE 19 Primers and summary of results of MA0317 genotyping. ExpectedCorrect Size Lane Template DNA Primers size(bp) Observed 1 1 KB ladderN/A N/A N/A 2 M3625 13092/17586 2368 yes 3 M3625 10871/14554 2966 yes 4M3625 16291/17887 2778 yes 5 M3625 16640/16509 1334 yes 6 M362513246/13095 2863 yes 7 1KB ladder N/A N/A N/A

TABLE 20 Sequence of primers used for genotyping MA0317. Primer SequenceSEQ ID NO 13092 ccacaccatagacttcagccttcttag 174 17586gcagtatgcaagtctcatgctg 175 10871 cgttcgctgtagcatacttagctat 176 14554ggctcttcattgagcttagaaccc 177 16291 aactggaccgatcttattcgt 178 17887actgcctcattgatggtggta 179 16640 cctcatcagctctggaacaacga 180 16509gtatgattgcggttatctgtcgc 181 13246 cctatggatgttgtaccatgcc 182 13095ccaatatcttgcagtccatcctcgtcgc 183

FIG. 38 shows the results of starch assay demonstrating starch degradingactivity in M3625. The assay was performed as described in copendingInternational Appl. No. PCT/US2011/039192, incorporated by referenceherein in its entirey.

Western Blot Protein Detection:

Anti-PflA, Anti-PflB, Anti-GA (AE9) and Anti-AdhE Antibodies:

In an effort to detect the presence of and help characterize a number ofenzymes engineered into the yeast strain, polyclonal antibodies wereproduced in rabbits at Lampire Biological Products, Pipersville, Pa.,against synthesized peptides with sequence similarity to the engineeredproteins. Table 11 depicts the peptides that were used as immunogens forthe rabbits:

TABLE 21 Immunogens used for antibody production Protein Immunogen SEQID NO Sf GA (AE9) intact purified protein 106 DNKNRYKINGNYKAGCNSGKKHIVESPQLSSRGGC CDHIDDNGQLTEEINRYTG Ba pflA CQNPDTWKMRDGKPVYYE 107GLTSSEENVENVAKIC Ba pflB WEGFTEGNWQKDIDVRDC 108 KQRDKDSIPYRNDFTECPECCNTITPDGLGRDEEEERIGN Ba AdhE DAKKKEEPTKPTPEEKLC 109 CKNLGVNPGKTPEEGVENCGSYGGNSVSGVNQAVN

For all of the synthesized peptides a terminal Cys was added forconjugation. Both the peptides and the purified GA protein wereconjugated to KLH prior to injection into the rabbit. A 50 day protocolwas used for antibody production with ELISA monitoring of the variousbleeds against the immunogen. After testing these polyclonal antibodiesin a Western blot against the lysate from the engineered yeast strains,serum from the positive rabbits was purified using a Protein G column.The purified antibodies were dialyzed into PBS, concentration wasdetermined by absorbance at 280 nm and the antibodies were used forfurther evaluation of the strains. Upon evaluation by SDS-PAGE, theantibodies appeared to be >90% pure.

Antibodies raised against the synthesized peptides were used in Westernblot detection of each engineered protein in cell extracts and culturesupernatants as described below.

Strain Growth Conditions:

Cells were plated from freezer stock on YPD (20 g/L peptone, 10 g/Lyeast extract, 20 g/L glucose) agar for 48 hours and used to inoculate25 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) in a 50mL culture tube. Cells were grown aerobically for 8 hours at 35° C. withshaking at 250 rpm, then lmL was removed to inoculate a sealed, CO2purged serum bottle containing 50 mL YPD (20 g/L peptone, 10 g/L yeastextract, 20 g/L glucose) with 7 mg/L ergosterol, 289 mg/L ethanol and544 mg/L Tween 80. These cultures were then grown anaerobicallyovernight (˜16 h) at 35° C. with shaking at 250 rpm. Cells wereharvested by centrifugation and washed with 25 mL deionized water. Theresulting wet cell pellets were used for Western blot detection of PflA,PflB and AdhE.

Aerobic cultures used to inoculate the serum bottles were returned tothe shaking incubator for an additional 40 hours. At the end ofincubation, cells were pelleted by centrifugation and the supernatantwas recovered and concentrated ˜10× using a 10 kDa molecular weightcut-off (MWCO) filter membrane. The resulting concentrates were used forWestern blot detection of extracellular AE9 glucoamylase.

Cell Lysis and Sample Preparation:

For Western blots of PflB and AdhE, cells were homogenized by mechanicaldisruption with 0.5 mm diameter beads and agitation at 4800 rpm in abead beater. 100 μL of wet cells were added to homogenization buffercontaining 1 mM phenylmethanesulfonylfluoride (PMSF), 2 mMdithiothreitol (DTT) and 1% dimethyl sulfoxide (DMSO) in 100 mM sodiumphosphate buffer pH 7.4. Cells were agitated for 6 cycles of 10 secondseach, cooling on ice between cycles. Cell debris was pelleted bycentrifugation and supernatant was recovered. 15 μL of the resultingsupernatant was added to 15 μL 2× concentrated SDS-PAGE sample bufferwith 50 mM DTT and loaded onto a 4-20% Tris-Glycine SDS-PAGE gel.

For Western blot detection of PflA, cells were lysed by adding 40 μL wetcells to 40 μL 2× concentrated SDS-PAGE sample buffer with 50 mM DTT.The mixture was then incubated at room temperature for 30 minutes,followed by heating at 100° C. for 2 minutes. Cells were pelleted bycentrifugation and 30 μL of the supernatant was loaded onto a 4-20%Tris-Glycine SDS-PAGE gel.

For AE9 analysis, 154, of concentrated aerobic culture supernatant wasadded to 15 μL, 2× concentrated SDS-PAGE sample buffer with 50 mM DTTand loaded onto a 4-20% Tris-Glycine SDS-PAGE gel.

Following gel electrophoresis, proteins were transferred to apolyvinylidine fluoride (PVDF) membrane and blocked overnight with Trisbuffered saline (TBS; 10 mM Tris, 150 mM sodium chloride pH 7.5)containing 2% weight by volume (w/v) bovine serum albumin (BSA). Theblocking solution was then removed, and primary peptide antibodies werediluted to approximately 2 μg/mL in Tris buffered saline with Tween 20(TBST; TBS with 0.1% v/v Tween 20) and added to each membrane. After a 1hour incubation, the primary antibody was discarded and the membrane waswashed for 3 periods of 5 minutes each in 10 mM Tris, 500 mM sodiumchloride, 0.1% Tween 20 pH 7.5 (THST). The secondary antibody, goatanti-rabbit with horseradish peroxidase label, was diluted 1:7500 inTBST, added to the blot and incubated for 1 hour. The secondary antibodywas then discarded and the blot was again washed with THST for 3 periodsof 5 minutes each. The wash solution was then discarded, enhancedchemiluminescence (ECL) substrate was added, and the blot was read by aseries of composite exposures using a gel imaging camera.

As shown in FIG. 39, for anti-PflA, PflB and AdhE primary antibodies,bands of approximately the correct molecular weight were detected ineach experimental strain, whereas no band was detected in the backgroundcontrol strain (M2390). For anti-AE9, bands were detected in strainsengineered to express the protein (M3625 and M3680) but were absent inother strains. See FIG. 39. There appeared to be two distinct bands forPflB, which may indicate oxygenic cleavage of the protein due to aerobiccell lysis conditions.

Pyruvate Formate Lyase Activity Assay:

Pyruvate formate lyase (PflB) is activated in the absence of oxygen byPfl activase (PflA) and catalyzes the reaction of pyruvate and CoA toformate and acetyl-CoA. The activity of PflB was measured in cellextracts by measuring formate production when extracts were added to areaction mixture containing pyruvate, CoA and DTT.

Strain Growth Conditions:

Cells were plated from freezer stock on YPD (20 g/L peptone, 10 g/Lyeast extract, 20 g/L dextrose) agar for 48 hours and used to inoculate25 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) in a50 mL culture tube. Cells were grown aerobically for 8 hours at 35° C.with shaking at 250 rpm, then lmL was removed to inoculate a sealed, CO2purged serum bottle containing 50 mL YPD (20 g/L peptone, 10 g/L yeastextract, 20 g/L dextrose) with 7 mg/L ergosterol, 289 mg/L ethanol and544 mg/L Tween 80. These cultures were then grown anaerobicallyovernight (˜16 h) at 35° C. with shaking at 250 rpm. Cells wereharvested by centrifugation and washed with 25 mL deionized water in ananaerobic chamber.

Cell Lysis and Sample Preparation:

Cells were homogenized in an anaerobic chamber by mechanical disruptionwith 0.5 mm diameter beads and agitation at 4800 rpm in a bead beater.100 μL of wet cells were added to homogenization buffer containing 1 mMPMSF, 2 mM DTT and 1% DMSO in 100 mM sodium phosphate buffer pH 7.4.Cells were agitated for 6 cycles of 10 seconds each, cooling on icebetween cycles. Cell debris was pelleted by centrifugation at 14,100×gfor 10 minutes and supernatant was recovered and clarified by filtrationthrough a 0.22 μm filter membrane. The resulting extract was useddirectly in the activity assay.

Pfl Activity Assay:

A 2× concentrated assay substrate mixture consisted of 20 mM sodiumpyruvate, 0.11 mM CoA and 20 mM DTT. Reagents were weighed out, broughtinto an anaerobic chamber and added to 10 mL of 100 mM sodium phosphatebuffer pH 7.4 which had been thoroughly degassed. 100 μL of cell extractwas added to 100 μL of the concentrated assay mixture and incubated atambient temperature (˜29° C.) for 30 minutes. Samples were then removedfrom the anaerobic chamber and heated in a heating block at 100° C. for90 seconds followed by cooling on ice to precipitate protein.Precipitate was removed by centrifugation at 15,000×g for 10 minutes.The resulting supernatant was analyzed for formate concentration usingthe formic acid assay kit available from Megazyme International Ireland,Bray, Co. Wicklow, Ireland.

Remaining cell extracts were diluted 1:8 in 100 mM sodium phosphatebuffer pH 7.4 and assayed for total protein content using the BCA totalprotein determination method. Formate concentrations of the Pfl assaysamples were normalized to the total protein concentration of thesample.

As shown in FIG. 40, experimental strains with engineered Pfl activity(M3465, M3625, M3679, and M3680) showed significantly higher amounts offormate present after incubation with the reaction mixture than thebackground control strain (M2390).

Alcohol Dehydrogenase E (AdhE) Enzymatic Activity Assays

AdhE is an intracellular bi-functional enzyme catalyzing the formationof ethanol from acetyl-CoA by way of acetaldehyde as an intermediate.This is accomplished by an acetaldehyde dehydrogenase activity and analcohol dehydrogenase activity working in series. Saccharomycescerevisiae strains have native alcohol dehydrogenase (Adh) activity; theintent of these activity assays is to show that Adh activity is retainedby the engineered strains, and an additional acetaldehyde dehydrogenaseactivity (from AdhE) is present.

Alcohol Dehydrogenase Activity:

As mentioned above, Bifidobacterium adolecentis bifunctional alcoholdehydrogenase (AdhE) has 2 primary functions. One function is theconversion of acetaldehyde to ethanol. This reversible reaction utilizesNADH as a cofactor. In order to evaluate the presence of this enzyme andensure that it has the desired activity, an assay was developed toevaluate the reverse reaction in which ethanol is converted toacetaldehyde. See FIG. 49. The rate of the reaction is monitored by NADHabsorbance at 340 nm.

Strain Growth Conditions:

Strains were patched from freezer stock onto a YPD (20 g/L peptone, 10g/L yeast extract, 20 g/L dextrose) agar plate and incubated overnightat 35° C. From that plate, 50 mL shake tubes with 25 mL YPD (20 g/Lpeptone, 10 g/L yeast extract, 20 g/L dextrose) were inoculated andincubated at 35° C., 250 rpm overnight. The cultures were centrifuged at5000 rpm×5 min at 4 C, washed with deionized (DI) water and centrifugedat 5000 rpm×5 min at 4 C, washed a second time with DI water andcentrifuged at 5000 rpm×5 min at 4 C, and then put on ice.

Cell Lysis and Sample Preparation:

100 μL of wet cell pellet was pipetted into a Zymo Research BashingBead0.5 mm Tubes along with 500 μL 100 mM Na2PO4, 2.5 mM MgCl2, 0.5 mM CaC12pH 7.4 buffer and 6 μL 100 mM phenylmethylsulfonyl fluoride (PMSF). Thecells were lysed by mechanical disruption using a MP FastPrep-24 set torun at 4.0 m/s for 10 seconds three times with cooling on ice for 10seconds between each run. This was repeated three times with chilling onice for one minute in between each run. Each tube was then centrifugedfor 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. Thesupernatant was removed and transferred to 2 mL tubes. 1 μL of NewEngland Biolabs DNAse I was added to each tube. The tubes were invertedand placed into an incubator set at 37° C. for 30 min. The tubes wereremoved from the incubator and the samples were transferred to 0.22 μmfilter centrifuge tubes which were centrifuged for 2 min at 10,000 rpm.50 μL of sample was pulled and diluted with 450 μL 100 mM Na₂PO₄ pH 7.4in separate sample tubes and then placed on ice.

Alcohol Dehydrogenase Activity Assay:

The assay used to determine alcohol dehydrogenase activity of AdhE wasadapted from the method of Vallee, B. L. and Hoch, F. L., Proc Natl AcadSci USA (1955) 41(6): 327-338. 100 μL 0.1M Na4P2O7 pH 9.6 buffer, 32 μL2M ethanol, and 1.664, 0.025M NAD⁺ were added to each well in a 96 wellplate. Once the lysate was added to the reaction mixture, the totalvolume of reaction was equivalent to 153.66 μL resulting in finalconcentrations of 65 mM Na₄P₂O₇ pH 9.6 buffer, 416.5 mM ethanol, and0.27 mM NAD⁺. To begin the reaction, 20 μL of 1:10 diluted lysate waspipetted into each well and the absorbance at 340 nm was observed andrecorded over 1.7 min using Spectramax M2 and Softmax software. Eachsample was done in duplicate to ensure reproducibility. ThermoScientific's BCA Protein Assay Kit was used to measure total proteinconcentration from the lysate generated. This data was used to normalizethe data generated from the actual reaction during analysis.

TABLE 22 Data for alcohol dehydrogenase activity assay Average activity(μmol Standard % p- NADH/min/mg) Deviation % CV Change value M2390 59915.6 2.6 M3465 659 16.2 2.5 10 0.1942 M3625 999.5 132 13.2 66.9 0.0108M3679 755 82 10.9 26 0.0603 M3680 698 29.7 4.3 16.5 0.0996 Alcoholdehydrogenase activities of engineered strains. p-value was based on aone-tailed T-test.

The background strain, M2390, performed as expected in this assay.Although it did not have AdhE engineered into its genome, it stillexpressed wild-type alcohol dehydrogenase and thus was active in thealcohol dehydrogenase assay. Other strains with AdhE engineered intotheir genomes should have expressed the bi-functional enzyme and shouldhave been more active given the total protein concentration was equal ineach sample used in the assay. With a p-value of <0.05, M3625demonstrated a statistically significant higher activity than thebackground strain. However, the other strains have a p-value>0.05indicating that they are within error of the background strain eventhough there was an increase in activity as shown by the % change overthe background activity. See Table 22. After normalizing the proteinconcentrations, a graphical representation of the data shows that eachstrain was more active than the background strain M2390 during a 1.7minute reaction period. FIG. 41 shows the activity of each strainplotted in μmol NADH/mg total protein vs. time during a 1.7 minutereaction.

Based on these results, the assay showed alcohol dehydrogenase activityin all strains. However, M2390 is less active and slower at convertingNAD⁺ to NADH than the other strains indicating that the engineered AdhEis present in each strain and it appears to be functioning properly.

Acetaldehyde Dehydrogenase Activity:

The second activity of AdhE is the reversible reaction convertingacetaldehyde to acetyl coenzyme A. This activity is not native toSaccharomyces cerevisiae strains, and should only be present in theengineered strains. In order to evaluate the presence of this enzyme andensure that it has the desired activity, an assay was developed tomeasure the conversion of acetaldehyde to acetyl CoA by AdhE. The rateof the reaction is monitored by NADH absorbance at 340 nm. A diagram ofthe reaction is provided in FIG. 50.

Strain Growth Conditions:

Strains were patched from a freezer stock onto a YPD (20 g/L peptone, 10g/L yeast extract, 20 g/L dextrose) agar plate and incubated overnightat 35° C. From that plate, 50 mL shake tubes with 25 mL YPD (20 g/Lpeptone, 10 g/L yeast extract, 20 g/L dextrose) were inoculated andincubated at 35° C., 250 rpm overnight. The cultures were centrifuged at5000 rpm×5 min at 4° C., washed with DI water and centrifuged at 5000rpm×5 min at 4 C, washed a second time with DI water and centrifuged at5000 rpm×5 min at 4° C., and then put on ice.

Cell Lysis and Sample Preparation:

100 μL of wet cell pellet was pipetted into a Zymo Research BashingBead0.5 mm Tubes along with 500 μL 100 mM Na₂PO₄, 2.5 mM MgCl₂, 0.5 mM CaCl₂pH 7.4 buffer and 64, 100 mM phenylmethylsulfonyl fluoride (PMSF). Thecells were lysed by mechanical disruption using a MP FastPrep-24 set torun at 4.0 m/s for 10 seconds three times with cooling on ice for 10seconds between each run. This was repeated three times with chilling onice for one minute in between each run. Each tube was then centrifugedfor 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. Thesupernatant was removed and transferred to 2 mL tubes. 1 μL of NewEngland Biolabs DNAse I was added to each tube. The tubes were invertedand placed into an incubator set at 37° C. for 30 min. The tubes wereremoved from the incubator and the samples were transferred to 0.22 μmfilter centrifuge tubes which were centrifuged for 2 min at 10,000 rpmusing the Eppendorf centrifuge 5424. 50 μL of sample was pulled anddiluted with 450 μL 100 mM Na₂PO₄ pH 7.4 in separate sample tubes andthen were placed on ice.

Acetaldehyde Dehydrogenase Activity Assay:

800 μL 50 mM Na₄P₂O₇ pH 9.6, 50 μL 0.025M NAD+, 50 μL 1M acetaldehyde,and 50 μL 1:10 diluted lysate were added to a Plastibrand microUV-cuvette. The cuvette was placed into a Shimadzu UV-1700 set to readabsorbance at 340 nm. 50 μL of 2 mM CoA were pipetted into the cuvettewhich was then mixed by gently pipetting the contents of the cuvette andthe absorbance was monitored for 5 minutes. The resulting finalconcentrations of each reagent were 40 mM Na₄P₂O₇ pH 9.6, 1.25 mM NAD⁺,50 mM acetaldehyde, and 0.1 mM CoA. Each sample was done in duplicate toensure reproducibility. Thermo Scientific's BCA Protein Assay Kit wasused to measure total protein concentration from the lysate generated.This data was used to normalize the data generated from the actualreaction during analysis.

Data for alcohol dehydrogenase (acetaldehyde dehydrogenase activity)assay is shown in Table 23. Note the lysate used in this assay was thesame lysate used in the alcohol dehydrogenase assay detailed in theprevious section.

TABLE 23 Acetaldehyde Dehydrogenase Activity Average activity (μmolStandard NADH/min/mg) Deviation % CV p-value M2390 0 0 0.0 M3465 163 2414.7 0.0054 M3625 115 7.07 6.2 0.0009 M3679 106 9.97 9.4 0.0022 M3680177 7.07 4.0 0.0004 Acetaldehyde dehydrogenase activity. p-value wasbased on a one-tailed T-test.

The background strain, M2390, performed as expected in this assay. Thewild-type strain should have no acetaldehyde dehydrogenase activity, asdemonstrated by this assay. The other strains with AdhE engineered intotheir genomes should have expressed the protein and had acetaldehydedehydrogenase activity. This activity was observed in all of theengineered strains (M3465, M3625, M3679, and M3680) with minimal errorand a p-value of <0.05.

Formate Dehydrogenase Activity

In strains M3465, M3625, M3679, and M3680 formate dehydrogenase wasknocked out of the genome in the hopes to balance redox with the variousengineering steps that were undertaken. The background strain, M2390,should have the gene intact. To ensure that the native Saccharomycescerevisiae formate dehydrogenase gene was removed, an enzymatic assaywas developed. Formate dehydrogenase catalyzes the conversion of formateto carbon dioxide at the expense of NAD⁺.Formate+NAD⁺→CO2+NADH  FDH

Enzymatic activity can be monitored by measuring NADH formation at 340nm.

Strain Growth Conditions:

M2390, M3465, M3625, M3679, and M3680 were patched from a freezer stockonto a YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) agarplate and incubated overnight at 35° C. From that plate, 50 mL shaketubes with 25 mL YPD+24 mM Sodium Formate were inoculated and incubatedat 35° C., 250 rpm overnight (20 g/L peptone, 10 g/L yeast extract, 20g/L dextrose). The cultures were centrifuged at 5000 rpm×5 min at 4° C.,washed with DI water and centrifuged at 5000 rpm×5 min at 4° C., washeda second time with DI water and centrifuged at 5000 rpm×5 min at 4 C,and then put on ice.

Cell Lysis and Sample Preparation:

100 μL of wet cell pellet was pipetted into a Zymo Research BashingBead0.5 mm Tubes along with 500 μL 100 mM Na₂PO₄, 2.5 mM MgCl₂, 0.5 mM CaCl₂pH 7.4 buffer and 64, 100 mM phenylmethylsulfonyl fluoride (PMSF). Thecells were lysed by mechanical disruption using a MP FastPrep-24 set torun at 4.0 m/s for 10 seconds three times with cooling on ice for 10seconds between each run. This was repeated three times with chilling onice for one minute in between each run. Each tube was then centrifugedfor 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. Thesupernatant was removed and transferred to 2 mL tubes. 1 μL of NewEngland Biolabs DNAse I was added to each tube. The tubes were invertedand placed into an incubator set at 37° C. for 30 min. The tubes wereremoved from the incubator and the samples were transferred to 0.22 μmfilter centrifuge tubes which were centrifuged for 2 min at 10,000 rpmusing the Eppendorf centrifuge 5424.

Formate Dehydrogenase Activity Assay:

800 μL 62.5 mM K₂PO₄ pH 7.0, 50 μL 40 mM NAD+, and 50 uL 1M SodiumFormate were added to a Plastibrand micro UV-cuvette. The cuvette wasplaced into a Shimadzu UV-1700 set to read absorbance at 340 nm andblanked. 100 μL of undiluted lysate sample were pipetted into thecuvette which was then mixed by gently pipetting the contents of thecuvette and the absorbance was monitored for 2.5 minutes. The resultingfinal concentrations of each reagent were 50 mM Potassium Phosphate, 2mM NAD+, and 0.05M Sodium Formate. Each sample was done in duplicate toensure reproducibility.

TABLE 24 Fdh activity of engineered strains Average μmol StandardNADH/min Deviation % CV M3631 0.03 0.00269 0.865 M2390 0.008 0 0 M3465 00 0 M3625 0 0 0 M3679 0 0 0 M3680 0 0 0

As shown in Table 24, the FDH knockout strains (M3465, M3625, M3679, andM3680) did not exhibit any formate dehydrogenase activity. Thebackground strain, M2390, had minimal activity. The positive controlstrain, M3631, which overexpresses FDH was active and produced asignificant amount of NADH that was observed and recorded.

AE9 Glucoamylase Activity Assay:

Saccharomycopsis fibuligera GLUT glucoamylase (AE9) produces glucosefrom starch.

Extracellular AE9 glucoamylase activity on raw corn starch was assayedto determine the presence of glucoamylase activity in aerobic culturesupernatants of engineered strains. Cells were grown aerobically,removed by centrifugation, and the resulting supernatant was assayed foractivity and compared to supernatant from strain M2390, which does notcontain AE9.

Cell Growth Conditions:

Cells were plated on YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/Ldextrose) agar for 48 hours and used to inoculate 25 mL YPD (20 g/Lpeptone, 10 g/L yeast extract, 20 g/L glucose) in a 50 mL culture tube.Cells were grown aerobically for 48 hours at 35° C. with shaking at 250rpm. After 48 hours, cells were removed via centrifugation and thesupernatant was recovered.

Sample Preparation:

The recovered aerobic culture supernatant was clarified by filtrationthrough a 0.22 μm filter membrane and concentrated ˜10× using a 10 kDamolecular weight cut-off filter. The retained concentrate was thenanalyzed for AE9 concentration via a phenyl reverse phase (phenyl-RP)HPLC method developed in-house using purified AE9 as a standard. Sampleswere diluted to an AE9 concentration of 50 m/mL and used directly in theactivity assay.

Glucoamylase Activity Assay:

A 2.2% (weight by volume) corn starch solution was made up in 50 mMsodium acetate buffer pH 5.0. In a 96-well assay plate, 50 μL ofsupernatant (adjusted to 50 m/mL AE9 concentration) was added to 450 μL2.2% starch. The plate was incubated at room temperature withoutshaking, and 50 μL of sample was taken at 1, 2, 5, 10, 30, 120 and 210minutes. Wells were mixed by pipette aspiration after initial enzymeaddition, as well as at each sampling thereafter. Samples were analyzedvia 3,5-dinitrosalicylic acid (DNS) method to determine reducing sugars.

As shown in FIG. 42, the aerobic culture supernatants of M3625 and M3680showed similar activity on raw corn starch, as measured by DNS analysis(Somogyi, M., Notes on Sugar Determination, JBC (200)45 (1952)).Amylolytic activity of M2390 supernatant was negligible in this assay.

The above data show that pflA, pflB and AdhE are present in strain M3625and have the proper activity. Fdh activity, seen in the backgroundstrain as well as in the positive control when fdh was overexpressed,was not present in the engineered strains indicating that this gene wasknocked out successfully.

Example 9

The following example demonstrates the ethanol yield of theSaccharomyces cerevisiae strain M3624. The genotype of strain M3624 is:Δgpd1::GPD2-B. adolescentis pflA/pFlB/adhEΔgpd2::GPD 1-B. adolescentispflA/pflB/adhE Δfdh1 Δfdh2::B. adolescentis pflA/pflB/adhE. Strain M3624was created according to the same methods employed above in Example 8.Detailed molecular maps for strain M3624 are shown in FIGS. 43A-D. FIG.43A shows insertion at the GPD1 locus; GPD2 expressed from the GPD1promoter; PFK2t-PFK2 terminator; HXT2t-HXT2 terminator; pADH1-ADH1promoter; PDC1 term-PDC1 terminator; FBA1 term-FBA1 terminator;pTPI1-TPI1 promoter; Scer ENOL ter-ENOL terminator. FIG. 43B showsinsertion at the GPD2 locus; GPD1 expressed from the GPD2 promoter; TDH3term-TDH3 terminator; pPFK1-PFK1 promoter; HXT2t-HXT2 terminator; PDC1term-PDC1 terminator; pADH1-ADH1 promoter; S. cer ENOL ter-ENOLterminator; FBA1 term-FBA1 terminator; pTPI1-TPI1 promoter. FIG. 43Cshows deletion of the FDH1 gene; flanking regions to create deletion ofFDH1. FIG. 43D shows insertion at the FDH2 locus; pPFK1-PFK1 promoter,S. cer ENOL ter-ENOL terminator; pADH1-ADh1 promoter; FBA1 term-FBA1terminator; PDC1 term-PDC1 terminator; pTPI-TPI1 promoter.

The data shown in FIG. 44 demonstrates that a 3.4% ethanol yieldincrease is obtained through reduction of glycerol and production offormate. M2390 is the control strain, M3515 and M3624 are engineeredwith the genotype Δgpd1::GPD2-B. adolescenits pflA/pflB/adhEΔgpd2::GPD1-B. adolescentis pflA/PFlB/adhE fdh1Δfdh2Δ::B. adolescentispflA/pflB/adhE. M3027 is engineered with the genotype Δgpd1Δgpd2::GPD1-B. adolescentis pflA/PFlB/adhE fdh1Δfdh2Δ::B. adolescentispflA/pflB/adhE. Panel A shows measurement of formate concentration,panel B shows measurement of glycerol concentration, and panel C showsmeasurement of ethanol concentration.

Both M3515 and M3624 have been engineered at 4 separate loci. The GPD1gene is expressed from the GPD2 promoter and the GPD2 gene is expressedfrom the GPD1 promoter, the FDH1 and FDH2 genes have been deleted.Additionally, the B. adolescentis pflA, pflB and adhE genes areexpressed as shown in FIGS. 43A, B, and D.

Example 10

The following example demonstrates the ethanol yield of theSaccharomyces cerevisiae strains M3465 and M3469. The genotype of strainM3465 is: Δgpd2:: B. adolescentis pflA/pflB/adhE Δfdh1Δ fdh2:: B.adolescentis pflA/pflB/adhE. The genotype of strain M3469 is: Δgpd1:: B.adolescentis pflA/pflB/adhE fdh1 Δ fdh2Δ:: B. adolescentispflA/pflB/adhE. Strains M3465 and M3469 were created according to thesame methods employed above in Example 8. Detailed molecular maps ofstrains M3465 and M3469 are shown in FIGS. 45 A-C and 46 A-C,respectively. FIG. 45A shows insertion at the GPD2 locus; pPFK1-PFK1promoter; HXT2t-HXT2 terminator; PDC1 term-PDC1 terminator; pADH1-ADH1promoter; S. cer ENOL ter-ENOL terminator; FBA1 term-FBA1 terminator;pTPI1-TPI1 promoter. FIG. 45B shows deletion of the FDH1 gene; flankingregions to create deletion of FDH1. FIG. 45C shows insertion at the FDH2locus; pPFK1-PFK1 promoter; S. cer ENOL ter-ENOL terminator; pADH1-ADh1promoter; FBA1 term-FBA1 terminator; PDC1 term-PDC1 terminator;pTPI-TPI1 promoter. FIG. 46A shows insertion at the GPD1 locus;pPFK1-PFK1 promoter; HXT2t-HXT2 terminator; PDC1 term-PDC1 terminator;pADH1-ADH1 promoter; S. cer ENO1 ter-ENO1 terminator; FBA1 term-FBA1terminator; pTPI 1-TPI1 promoter. FIG. 46B shows deletion of the FDH1gene; flanking regions to create deletion of FDH1. FIG. 46C showsinsertion at the FDH2 locus; pPFK1-PFK1 promoter, S. cer ENOL ter-ENO1terminator; pADH1-ADh1 promoter; FBA1 term-FBA1 terminator; PDC1term-PDC1 terminator; pTPI-TPI1 promoter.

This example demonstrates that the ethanol yield increase is dependenton the level of glycerol reduction. Fermentation of 30% solids corn mashby M3465, which contains a deletion of the GPD2, FDH1 and FDH2 genes andexpression of B. adolescentis pflA, pflB and adhE genes from the GPD2and FDH2 loci, results in a 1.5% increase in ethanol titer. As shown inFIG. 47, fermentation of 30% solids corn mash by M3469, which contains adeletion of the GPD1, FDH1 and FDH2 genes and expression of B.adolescentis pflA, pflB and adhE genes from the GPD1 and FDH2 loci,results in a 2.5% increase in ethanol titer. M2390 is the control parentstrain. As shown in FIG. 48, fermentation of corn mash by M3465 andM3469 results in ˜15% lower glycerol and 30% lower glycerol levelsrespectively. M2390, represented in FIGS. 47 and 48, is the controlstrain.

Example 11

An alternative way to reduce glycerol formation is through deletion ofthe glycerol-3-phosphate phosphatase (GPP) genes. Saccharomyces containstwo copies of these genes, GPP1 and GPP2. The data below demonstratesthat expression of B. adolescentis pflA, pflB and adhE in backgroundscontaining deletions of FDH1, FDH2 and either GPP1 or GPP2 results indecreased glycerol formation (FIG. 53; strain comparison, min buffmedium, glucose 40 g/L, anaerobic fermentations, 35° C.—72 hr.) andincreased ethanol yield (FIG. 54; strain comparison, min buff medium,glucose 40 g/L, anaerobic fermentations, 35° C.—72 hr.). Production offormate was also observed. See FIG. 55 (strain comparison, min buffmedium, glucose 40 g/L, anaerobic fermentations, 35° C.—72 hr.).

The strains engineered to measure the glycerol formation, ethanol yield,and formate production were the Saccharomyces cerevisiae strains M3297,TB655, and TB656. Strains M3297, TB655, and TB656 were created accordingto the same methods employed above in Example 8. The genotype of strainM3297 is: Δfdh1Δfdh2::pflA/pflB/adhE. This strain contains only deletionin the FDH genes plus expression of pflA, pflB and AdhE. The genotype ofstrain TB655 is: Δfdh1Δfdh2::pflA/pflB/adhEΔgpp1::pflA/pflB/adhE. Thisstrain contains deletion in the FDH genes, expression of pflA, pflB andAdhE, and deletion of GPP1. See FIG. 51. The genotype of strain TB656is: Δfdh1Δfdh2::pflA/pflB/adhEΔgpp2::pflA/pflB/adhE. This straincontains deletion in the FDH genes, expression of pflA, pflB and AdhEand deletion of GPP1. See FIG. 52.

The amount of ethanol, glycerol, and formate produced by strains TB655and TB656 was measured using the methods described above. Compared tothe control strain M3297, strains TB655 and TB656 demonstratedstatistically significant changes in the amount of ethanol, glycerol,and formate produced. Relative to strain M3297, strain TB655 (gpp1mutant) demonstrated a 1.3% increase in ethanol titer, 10% reduction inglycerol, and 100% more formate produced, whereas strain TB656 (gpp2mutant) demonstrated a 0.95% increase in ethanol titer, 6.1% reductionin glycerol formation, and 100% more formate produced. These resultsdemonstrate the novel combination of GPP mutation with a metabolicengineering solution to balance redox during anaerobic growth.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A recombinant microorganism comprising: (a) adeletion of one or more native enzymes that function to produce glyceroland/or regulate glycerol synthesis, wherein said one or more enzymes isencoded by a glycerol-3-phosphate dehydrogenase (gpd1),glycerol-3-phosphate dehydrogenase (gpd2) glycerol 3-phosphatase 1′,glycerol 3-phosphatase 2 or fps1 polynucleotide; and (b) one or morenative and/or heterologous enzymes that function in one or moreengineered metabolic pathways to convert a carbohydrate source toethanol, wherein one of said metabolic pathways comprises conversion ofpyruvate to acetyl-CoA and formate by a pyruvate formate lyase, whereinone of said metabolic pathways comprises conversion of acetyl-CoA toethanol by an acetaldehyde dehydrogenase, alcohol dehydrogenase, or abifunctional acetaldehyde/alcohol dehydrogenase, and wherein said one ormore native and/or heterologous enzymes is activated, upregulated ordownregulated.
 2. The recombinant microorganism of claim 1, wherein saidone or more native enzymes that function to produce glycerol is encodedby both a gpd1 polynucleotide and a gpd2 polynucleotide, and wherein oneof said engineered metabolic pathways comprises conversion of acetyl-CoAto ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, andfurther comprising a deletion of one or more native enzymes encoded byboth an fdh1 polynucleotide and an fdh2 polynucleotide.
 3. Therecombinant microorganism of claim 1, wherein said one or more nativeenzymes that function to produce glycerol is encoded by both a gpp1polynucleotide and a gpp2 polynucleotide, and wherein one of saidengineered metabolic pathways comprises conversion of acetyl-CoA toethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, andfurther comprising a deletion of one or more native enzymes encoded byboth an fdh1 polynucleotide and an fdh2 polynucleotide.
 4. Therecombinant microorganism of claim 1, wherein said one or more nativeenzymes that function to regulate glycerol synthesis is encoded by anfps1 polynucleotide, and wherein one of said engineered metabolicpathways comprises conversion of acetyl-CoA to ethanol by a bifunctionalacetaldehyde/alcohol dehydrogenase, and further comprising a deletion ofone or more native enzymes encoded by both an fdh1 polynucleotide and anfdh2 polynucleotide.
 5. The recombinant microorganism of claim 1,wherein said one or more native enzymes that function to regulateglycerol synthesis is encoded by an fps1 polynucleotide and said one ormore native enzymes that function to produce glycerol is encoded by botha gpd1 polynucleotide and a gpd2 polynucleotide, wherein one of saidengineered metabolic pathways comprises conversion of acetyl-CoA toethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, andfurther comprising a deletion of one or more native enzymes encoded byboth an fdh1 polynucleotide and an fdh2 polynucleotide.
 6. Therecombinant microorganism of claim 1, wherein said one or more nativeenzymes that function to produce glycerol is encoded by both a gpd1polynucleotide and a gpd2 polynucleotide and wherein one of saidengineered metabolic pathways comprises conversion of acetyl-CoA toethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, furthercomprising a native and/or heterologous gpd1 polynucleotide operablylinked to a native gpd2 promoter polynucleotide.
 7. The recombinantmicroorganism of claim 6, further comprising a deletion of one or morenative enzymes encoded by both an fdh1 polynucleotide and an fdh2polynucleotide.
 8. The recombinant microorganism of claim 1, whereinsaid one or more native enzymes that function to produce glycerol isencoded by both a gpd1 polynucleotide and a gpd2 polynucleotide and saidone or more native enzymes that function to regulate glycerol synthesisis encoded by an fps1 polynucleotide, and wherein one of said engineeredmetabolic pathways comprises conversion of acetyl-CoA to ethanol by abifunctional acetaldehyde/alcohol dehydrogenase, further comprising anative and/or heterologous gpd1 polynucleotide operably linked to anative gpd2 promoter polynucleotide.
 9. The recombinant microorganism ofclaim 8, further comprising a deletion of one or more native enzymesencoded by both an fdh1 polynucleotide and an fdh2 polynucleotide. 10.The recombinant microorganism of claim 2, wherein one of said engineeredmetabolic pathways comprises conversion of a carbohydrate source to oneor more sugar units by a saccharolytic enzyme.
 11. The recombinantmicroorganism of claim 3, wherein one of said engineered metabolicpathways comprises conversion of a carbohydrate source to one or moresugar units by a saccharolytic enzyme.
 12. The recombinant microorganismof claim 4, wherein one of said engineered metabolic pathways comprisesconversion of a carbohydrate source to one or more sugar units by asaccharolytic enzyme.
 13. The recombinant microorganism of claim 5,wherein one of said engineered metabolic pathways comprises conversionof a carbohydrate source to one or more sugar units by a saccharolyticenzyme.
 14. The recombinant microorganism of claim 6, wherein one ofsaid engineered metabolic pathways comprises conversion of acarbohydrate source to one or more sugar units by a saccharolyticenzyme.
 15. The recombinant microorganism of claim 7, wherein one ofsaid engineered metabolic pathways comprises conversion of acarbohydrate source to one or more sugar units by a saccharolyticenzyme.
 16. The recombinant microorganism of claim 8, wherein one ofsaid engineered metabolic pathways comprises conversion of acarbohydrate source to one or more sugar units by a saccharolyticenzyme.
 17. The recombinant microorganism of claim 9, wherein one ofsaid engineered metabolic pathways comprises conversion of acarbohydrate source to one or more sugar units by a saccharolyticenzyme.
 18. The recombinant microorganism of claim 1, wherein said oneor more native enzymes that function to produce glycerol is encoded by agpd2 polynucleotide, and wherein one of said engineered metabolicpathways comprises conversion of acetyl-CoA to ethanol by a bifunctionalacetaldehyde/alcohol dehydrogenase and one of said engineered metabolicpathways comprises conversion of a carbohydrate source to one or moresugar units by a saccharolytic enzyme, and further comprising a deletionof one or more native enzymes encoded by both an fdh1 polynucleotide andan fdh2 polynucleotide.